NO801018L - DIAGNOSTIC PROCEDURES. - Google Patents
DIAGNOSTIC PROCEDURES.Info
- Publication number
- NO801018L NO801018L NO801018A NO801018A NO801018L NO 801018 L NO801018 L NO 801018L NO 801018 A NO801018 A NO 801018A NO 801018 A NO801018 A NO 801018A NO 801018 L NO801018 L NO 801018L
- Authority
- NO
- Norway
- Prior art keywords
- cancer
- concentration
- dna
- protein
- bleomycin
- Prior art date
Links
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Classifications
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- 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
- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
- G01N33/57407—Specifically defined cancers
- G01N33/57411—Specifically defined cancers of cervix
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- 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
- G01N33/52—Use of compounds or compositions for colorimetric, spectrophotometric or fluorometric investigation, e.g. use of reagent paper and including single- and multilayer analytical elements
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- 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
- G01N33/5308—Immunoassay; Biospecific binding assay; Materials therefor for analytes not provided for elsewhere, e.g. nucleic acids, uric acid, worms, mites
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- 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
- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
- G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
- G01N33/57488—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites involving compounds identifable in body fluids
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- 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
- G01N33/6875—Nucleoproteins
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- 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
- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- G01N2333/46—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates
- G01N2333/47—Assays involving proteins of known structure or function as defined in the subgroups
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- 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
- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- G01N2333/46—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates
- G01N2333/47—Assays involving proteins of known structure or function as defined in the subgroups
- G01N2333/4701—Details
- G01N2333/4703—Regulators; Modulating activity
- G01N2333/4704—Inhibitors; Supressors
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- 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
- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- G01N2333/475—Assays involving growth factors
- G01N2333/50—Fibroblast growth factors [FGF]
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- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
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Description
Foreliggende oppfinnelse angår en fremgangsmåte for' diagnostisk påvisning av maligne neoplasmer. The present invention relates to a method for the diagnostic detection of malignant neoplasms.
Det er et klart behov for nye fremgangsmåter for påvisning av kreft. I mange tilfeller vil en tidlig diagnose av kreft i høy grad øke sjansene til å bevirke fullstendig remi-sjon av sykdommen. There is a clear need for new methods for detecting cancer. In many cases, an early diagnosis of cancer will greatly increase the chances of causing complete remission of the disease.
Det er i litteraturen beskrevet tidligere forsøk på å påvise nærvær av tumor-spesifikke komponenter, såsom hormoner og antigener, i blodet hos kreftpasienter. Slike forsøk har imidlertid stort sett vært uten gunstig resultat, og en praktisk, ikke-inngripende diagnostisk metode, som er basert på innholdet av en tumor-spesifikk serumkomponent har hittil stått som et uoppnåelig mål. Previous attempts to demonstrate the presence of tumor-specific components, such as hormones and antigens, in the blood of cancer patients have been described in the literature. However, such attempts have largely been without favorable results, and a practical, non-invasive diagnostic method, which is based on the content of a tumor-specific serum component, has so far stood as an unattainable goal.
Nylig er det funnet DNA-bindende proteiner i.serum hos pasienter med neoplasis, systemisk lupus erythematosus og andre inflammatoriske sykdommer (se f.eksi. FEBS Letters 92(2) : 211-213 (1978); Europ. J. Biochem. 71:1-8 (1976); Amer. J.. Med. 6_5: 437-445 (1978). Det-er imidlertid ikke frembrakt en egnet undersøkelsesmetode for slike serumproteiner, noe som vil være nødvendig for en praktisk diagnostisk fremgangsmåte. Videre ser det ut til at de tidligere beskrevne serumproteiner ikke oppviser den grad av selektivitet, som. er ønsket ved en screening-metode for kreft. Recently, DNA-binding proteins have been found in the serum of patients with neoplasia, systemic lupus erythematosus and other inflammatory diseases (see e.g. FEBS Letters 92(2) : 211-213 (1978); Europ. J. Biochem. 71 :1-8 (1976); Amer. J.. Med. 6_5: 437-445 (1978). However, no suitable examination method for such serum proteins, which will be necessary for a practical diagnostic procedure. Furthermore, it appears that the previously described serum proteins do not exhibit the degree of selectivity that. is desired by a screening method for cancer.
Foreliggende oppfinnelse gjør bruk av et inhibitor-serumprotein som er beskrevet i " The Pharmacologists" ( Abs.) 2_0(3):238 (1978). Sammendraget gir imidlertid ingen antydning av at proteinet er tilstéde i forskjellige mengder hos pasienter med kreft og hos kreftfrie pasienter. •Foreliggende oppfinnelse tilveiebringer således en generelt anvendelig fremgangsmåte for diagnostisk undersøkelse for påvisning av maligne neoplasmer i mennesker. Mere spesielt frembringer oppfinnelsen en diagnostisk fremgangsmåte for påvisning av forekomsten av maligne neoplasmer ved undersøkelse av humane serumprøver for nærvær av et spesifikt DNA-bindende protein. Fremgangsmåten er verdifull for tidlig påvisning av maligne sykdommer og som en indikator for sykdommens utvikling hos en kreftpasient som er under behandling. The present invention makes use of an inhibitor serum protein which is described in "The Pharmacologists" (Abs.) 2_0(3):238 (1978). However, the abstract gives no indication that the protein is present in different amounts in patients with cancer and in cancer-free patients. • The present invention thus provides a generally applicable method for diagnostic examination for the detection of malignant neoplasms in humans. More particularly, the invention provides a diagnostic method for detecting the presence of malignant neoplasms by examining human serum samples for the presence of a specific DNA-binding protein. The procedure is valuable for the early detection of malignant diseases and as an indicator of the development of the disease in a cancer patient who is undergoing treatment.
Foreliggende fremgangsmåte er basert på den oppdagelse at et visst DNA-bindende protein (heretter kalt inhibitor-protein X) er tilstede i vesentlig reduserte konsentrasjoner i serumet hos pasienter med kreft i forhold til kreft-frie pasienter. Ved å tilveiebringe en følsom prøve for dette serumprotein muliggjør foreliggende oppfinnelse en hurtig, ikke-inngripende og nøyaktig fremgangsmåte for påvisning av forekomsten av kreft. The present method is based on the discovery that a certain DNA-binding protein (hereafter called inhibitor protein X) is present in substantially reduced concentrations in the serum of patients with cancer compared to cancer-free patients. By providing a sensitive test for this serum protein, the present invention enables a rapid, non-invasive and accurate method for detecting the presence of cancer.
Nærværet av et bestemt DNA-bindende protein i humant serum ble oppdaget av de foreliggende oppfinnere under under-søkelse av den bleomycin-induserte nedbrytning av PM-2 DNA (Pseudomonas bakteriofag kovalent lukket sirkulær DNA). Iakt-tagelse av inhiberingen av denne nedbrytning av humant serum førte til studier over inhiberingens art og forsøk på å iden-tifisere inhibitorforbindelsen. The presence of a particular DNA-binding protein in human serum was discovered by the present inventors while investigating the bleomycin-induced degradation of PM-2 DNA (Pseudomonas bacteriophage covalently closed circular DNA). Observation of the inhibition of this degradation by human serum led to studies on the nature of the inhibition and attempts to identify the inhibitor compound.
De foreliggende oppfinneres hypotese, nemlig at inhiberingen av PM-2 DNA nedbrytningsreaksjonen skyldtes et serumprotein, ble bekreftet da behandling med pronase resulterte i tapet av inhibitorisk aktivitet. Behandlingen med DNAase 1 eller pankreatisk RNAase hadde ingen virkning på den inhibitoriske aktivitet av serumet. Sera fra hunder og katter var ikke inhibitorisk, hvilket heller ikke var tilfelle for humant albumin og immunoglobuliner. The present inventors' hypothesis, namely that the inhibition of the PM-2 DNA degradation reaction was due to a serum protein, was confirmed when treatment with pronase resulted in the loss of inhibitory activity. The treatment with DNAase 1 or pancreatic RNAase had no effect on the inhibitory activity of the serum. Sera from dogs and cats were not inhibitory, nor were human albumin and immunoglobulins.
Inhibitor-proteinet ble renset under ikke-denaturerende betingelser under anvendelse av molekylfiltre, dialyse og selv-kromatografi med "Sephadex". Rensning av proteinet ble vesentlig forbedret ved å binde proteinet til DNA, isolere DNA-protein komplekset fra det ubundne protein og deretter dissociere proteinet fra DNA ved behandling med urinstoff. Rensning ved SDS-polyakrylamid gel-elektroforese og isoelektrisk fokusering antas å resultere i en rensning på mer enn 2000 ganger. Isoe-, lektrisk fokuserende gel-eletroforese antyder at inhibitoren er et protein med en molekylvekt på ca. 64.000 dalton og en pl på 5,9. Dette protein er foreløbig her kalt inhibitor-protein The inhibitor protein was purified under non-denaturing conditions using molecular filters, dialysis and self-chromatography with Sephadex. Purification of the protein was significantly improved by binding the protein to DNA, isolating the DNA-protein complex from the unbound protein and then dissociating the protein from DNA by treatment with urea. Purification by SDS-polyacrylamide gel electrophoresis and isoelectric focusing is believed to result in more than 2000-fold purification. Isoelectric focusing gel electrophoresis suggests that the inhibitor is a protein with a molecular weight of approx. 64,000 daltons and a pl of 5.9. This protein is provisionally called inhibitor protein here
X. X.
Inhibitor-protein X utøver sin inhibitoriske virkning ved å binde til DNA. DNA-binding er blitt påvist ved agarose gel-elektroforese, isolering av DNA-proteinkomplekset, fluor-essenskjøling og sirkulære dikroisme studier. Inhibitor protein X exerts its inhibitory effect by binding to DNA. DNA binding has been demonstrated by agarose gel electrophoresis, isolation of the DNA-protein complex, fluorescein quenching and circular dichroism studies.
Ved undersøkelsen av arten av inhibitor-protein X viste det seg uventet at det var en signifikant forskjell i innholdet av dette protein i serum hos pasienter med kreft i forhold til serum hos kreft-frie pasienter. Basert på denne viktige oppfinnelse søkte man som et ledd i den foreliggende oppfinnelse etter en følsom og hurtig prøve for nærværet av inhibitor-protein X i humant serum. When investigating the nature of inhibitor protein X, it unexpectedly turned out that there was a significant difference in the content of this protein in the serum of patients with cancer compared to the serum of cancer-free patients. Based on this important invention, a sensitive and rapid test for the presence of inhibitor protein X in human serum was sought as part of the present invention.
PM-2 DNA har tidligere vært anvendt ved en spektrofoto-fluorometrisk prøve for bestemmelse av bleomycin biokjemisk aktivitet ( Cancer Res. 38:3322-3326 (1978)). Bleomycins virk-. ningsmekanisme synes å henge sammen med dets evne til å bryte ned DNA, og forminskelsen i binding av éthidiumbromid (2,7-diamino-10-etyl-9-fenyl-fenanthridiniumbromid) til PM-2 DNA indusert av bleomycin, som bestemt.ved fluoressens-spektrometri, kan derfor anvendes ved undersøkelse av bleomycinaktivitet. PM-2 DNA has previously been used in a spectrophotofluorometric assay for the determination of bleomycin biochemical activity (Cancer Res. 38:3322-3326 (1978)). Bleomycin's effect. mechanism appears to be related to its ability to degrade DNA, and the reduction in binding of ethidium bromide (2,7-diamino-10-ethyl-9-phenyl-phenanthridinium bromide) to PM-2 DNA induced by bleomycin, as determined by fluorescence spectrometry, can therefore be used when examining bleomycin activity.
Som anført ovenfor har det vist seg at inhibitor-protein X virker som en inhibitor for bleomycin-indusert nedbrytning av PM-2 DNA. Som følge herav kan den tidligere til måling av bleomycinaktiviteten anvendte fluorometriske prøve også anvendes til undersøkelse for konsentrasjonen av inhibitor-protein X i humant serum. Innholdet av inhibitor-protein X kan deretter anvendes til å forutsi sannsynligheten for kreft hos pasienter som undersøkes. As noted above, inhibitor protein X has been shown to act as an inhibitor of bleomycin-induced degradation of PM-2 DNA. As a result, the fluorometric sample previously used to measure bleomycin activity can also be used to examine the concentration of inhibitor protein X in human serum. The content of inhibitor protein X can then be used to predict the likelihood of cancer in patients being examined.
Foreliggende oppfinnelse anviser således en fremgangsmåte for påvisning av kreft hos mennesker der man: (a) tar en blodprøve fra en pasient som skal undersøkes med henblikk på kreft, The present invention thus prescribes a method for detecting cancer in humans where: (a) a blood sample is taken from a patient who is to be examined for cancer,
og fremgangsmåten karakteriseres ved at manand the procedure is characterized by the fact that one
(b) bestemmer konsentrasjonen av inhibitor-protein X i prøven fra trinn (a), og (c) sammenligner den i trinn (b) bestemte konsentrasjon med norm-konsentrasjonen av inhibitor-protein X i serum.hos kreft-frie pasienter, hvorved en signifikant reduksjon av konsentrasjonen av inhibitor-protein X i forhold til norm-konsentras jonen indikerer sannsynligheten for kreft. (b) determines the concentration of inhibitor protein X in the sample from step (a), and (c) compares the concentration determined in step (b) with the norm concentration of inhibitor protein X in the serum of cancer-free patients, whereby a significant reduction in the concentration of inhibitor protein X in relation to the norm concentration indicates the likelihood of cancer.
Den spesielle undersøkelsesmetode: som anvendes i trinn. The special survey method: which is used in stages.
(b) til bestemmelse av serumkonsentrasjonen av inhibitor-protein X er ikke vesentlig og den foreliggende oppfinnelse ligger (b) for determining the serum concentration of inhibitor protein X is not essential and the present invention lies
i sitt bredeste aspekt i oppdagelsen av at inhibitor-protein X konsentrasjonen (uansett hvordan den er bestemt) kan anvendes som et diagnostisk middel for en lang rekke humane krefttilfel-ler. in its broadest aspect in the discovery that inhibitor protein X concentration (however determined) can be used as a diagnostic tool for a wide range of human cancers.
En undersøkelsesmetode som har vist seg å være fordel-aktig er den PM-2 DNA fluoressens-prøve som er beskrevet i " Cancer Res." 38: 3322-3326 (1976). Anvendelse av denne prøve er basert på den inhibitoriske virkning av inhibitor-protein X. på den velkjente bleomycin-induserte nedbrytning av PM-2 DNA reaksjonen. Når denne prøve anvendes er det hensiktsmessig å uttrykke konsentrasjonen av inhibitor-protein X som en IC^q- ' verdi, der ICb,.U..er definert som den konsentrasjon av inhibitor-protein X som kreves for å inhibere 50% bleomycin-indusert nedbrytning av PM-2 DNA. Denne IC^Q-verdi kan deretter sammenlig-nes med norm IC^^-verdier som er knyttet til serum hos kreft-frie pasienter, og en signifikant stigning i IC^Q-verdien i forhold til norm-verdien vil indikere sannsynligheten for kreft. One screening method that has been shown to be advantageous is the PM-2 DNA fluorescence assay described in "Cancer Res." 38: 3322-3326 (1976). Application of this test is based on the inhibitory effect of inhibitor protein X on the well-known bleomycin-induced breakdown of the PM-2 DNA reaction. When this test is used, it is appropriate to express the concentration of inhibitor protein X as an IC^q value, where ICb,.U..is defined as the concentration of inhibitor protein X required to inhibit 50% of bleomycin induced degradation of PM-2 DNA. This IC^Q value can then be compared with norm IC^^ values which are linked to the serum of cancer-free patients, and a significant rise in the IC^Q value in relation to the norm value will indicate the probability of cancer .
I det følgende beskrives nærmere PM-2 DNA fluoressens-prøven for inhibitor-protein X: Det fremstilles først en.blanding av en serumprøve (f.- ■ eks.-en 50 yl prøve) fra en pasient som skal undersøkes og en oppløsning av bleomycin, PM-2 DNA og 2-mercaptoetanol i pH. 9,5 puffer. Karakteristisk blandes en 50 yl serumprøve med 450'yl av en oppløsning inneholdende 35 nM bleomycin, 8,3 yM PM-2 DNA og 25 mM 2-mercaptoetanol i pH 9,5 natriumboratpuffer (0,015M NaCl: 0,05M natriuborat). , ' In the following, the PM-2 DNA fluorescence test for inhibitor protein X is described in more detail: First, a mixture of a serum sample (e.g. ■ eg a 50 µl sample) from a patient to be examined and a solution of bleomycin, PM-2 DNA and 2-mercaptoethanol in pH. 9.5 puffs. Typically, a 50 µl serum sample is mixed with 450 µl of a solution containing 35 nM bleomycin, 8.3 µM PM-2 DNA and 25 mM 2-mercaptoethanol in pH 9.5 sodium borate buffer (0.015M NaCl: 0.05M sodium borate). , '
Ovenfor^ angitte blanding inkuberes deretter ved 3 7°C i 3 0 minutter The above mixture is then incubated at 37°C for 30 minutes
En oppløsning av ethidiumbromid i pH 12,1 denaturérings-puffer fremstilles deretter ved tilsetning av 0,1 ml av en ethidiumbromidoppløsning (22 yg ethidiumbromid pr. ml 12,1 denatureringspuffer) til 0,9 ml av en pH 12,1 denatureringspuffer. En passende denatureringspuf f er omfatter 0,09M Na^PO^; : 0,01M EDTA : 0,01M NaCl, innstilt på pH-verdi 12,1 med 0,15M NaOH. Til 1 ml av denne ethidiumbromidoppløsning settes en porsjon, fleks. 100 yl, av den inkuberte serumblanding fremstilt som angitt ovenfor. A solution of ethidium bromide in pH 12.1 denaturation buffer is then prepared by adding 0.1 ml of an ethidium bromide solution (22 µg ethidium bromide per ml of 12.1 denaturation buffer) to 0.9 ml of a pH 12.1 denaturation buffer. A suitable denaturing buffer f is comprises 0.09M Na^PO^; : 0.01M EDTA : 0.01M NaCl, adjusted to pH value 12.1 with 0.15M NaOH. To 1 ml of this ethidium bromide solution is added a portion, flex. 100 µl, of the incubated serum mixture prepared as indicated above.
Fluoressensen av ethidiumbromid: PM-2 DNA blandingen bestemmes med et spektrofotofluorometer ved 53 0 nm inngang og 590 nm emisjon. Fluoressens større enn bakgrunnen forårsakes av bindingen av ethidiumbromid til PM-2 DNA, slik at en endring i fluoressens i forhold til en kontrollreaksjon (identisk med testprøve bortsett fra at den ikke inneholder bleomycin) til-later at graden av bleomycin-indusert nedbrytning av PM-2 DNA kan kvantifiseres. Utfra fluoressensverdiene kan den prosentuelle inhibering av PM-2 DNA nedbrytning som forårsakes av nærværet av inhibitor-protein X i serumet lett bestemmes. The fluorescence of the ethidium bromide:PM-2 DNA mixture is determined with a spectrophotofluorometer at 530 nm input and 590 nm emission. Fluorescence greater than background is caused by the binding of ethidium bromide to PM-2 DNA, so that a change in fluorescence relative to a control reaction (identical to test sample except that it does not contain bleomycin) allows the degree of bleomycin-induced degradation of PM -2 DNA can be quantified. From the fluorescence values, the percentage inhibition of PM-2 DNA degradation caused by the presence of inhibitor protein X in the serum can be easily determined.
Konsentrasjonen av inhibitor-protein X i serumprøven bestemmes deretter ved dén konvensjonelle Lowry-metode (folin-fenol-reagens) som beskrevet i " J. Biol. Chem." 193:265-275 The concentration of inhibitor protein X in the serum sample is then determined by the conventional Lowry method (folin-phenol reagent) as described in "J. Biol. Chem." 193:265-275
(.19 51). (.19 51).
Ved å avbilde den prosentuelle inhibering av PM-2 DNA nedbrytning mot logaritmen til konsentrasjonen av inhibitor-protein X kan. IC^g-verdien for den bestemte serumprøve lett bestemmes.. By plotting the percentage inhibition of PM-2 DNA degradation against the logarithm of the concentration of inhibitor protein X can. The IC^g value for the particular serum sample is easily determined..
Når først IC,. Q-verdien for en gitt pasient er beregnet kan denne verdi anvendes direkte til å forutsi sannsynligheten hos kreft, hos en slik pasient. Omfattende studier på friske When first IC,. The Q-value for a given patient has been calculated, this value can be used directly to predict the probability of cancer in such a patient. Extensive studies on healthy subjects
(kreft-frie) pasienter og pasienter med mange forskjellige maligne neoplasmer har vist at inhibitor-protein X finnes i signifikant lavere mengder i serum hos pasienter med kreft. IC|-Q-verdien finnes derfor hos kreftpasienter å være signifikant forhøyet i forhold til norm-veriden for friske individer. Denne klare differensiering i ICj-g-verdiene gir en enkel og nøyaktig basis for påvisning av nærvær av kreft og er kjernen i foreliggende oppfinnelse. (cancer-free) patients and patients with many different malignant neoplasms have shown that inhibitor protein X is found in significantly lower amounts in the serum of patients with cancer. The IC|-Q value is therefore found in cancer patients to be significantly elevated compared to the norm for healthy individuals. This clear differentiation in the ICj-g values provides a simple and accurate basis for detecting the presence of cancer and is the core of the present invention.
Den nøyaktige IC^Q-verdi som oppnås fra blodprøver hos kreft-frie pasienter vil variere i en viss grad med det bestemte individ. Hos pasienter som hittil har. vært prøvet har den gjennomsnittlige IC^^-verdi for kreft-frie pasienter vist seg å være 90, 2- 11,0 pg/ml (signifikant ved 0,001 nivået). Den høyeste registrerte ICj-g-verdi for en frisk pasient har vært 120 yg/ml. The exact IC^Q value obtained from blood samples of cancer-free patients will vary to some extent with the particular individual. In patients who have previously been tested, the mean IC^^ value for cancer-free patients has been found to be 90.2-11.0 pg/ml (significant at the 0.001 level). The highest recorded ICj-g value for a healthy patient has been 120 yg/ml.
Hos kreftpasienter varierte de oppnådde IC[-Q-verdier med den involverte krefttype,, men i alle tilfelle har til nu verdien vært signikant høyere enn hos kreft-frie individer. Den laveste IC5Q-verdi til nu for en kreftpasient har vært 270 vig/ml. Imidlertid indikerer en IC,-_-verdi over ca. 200 dog en stor sannsynlighet for. at pasienten har en malign neo-plasme av en eller annen type. In cancer patients, the obtained IC[-Q values varied with the type of cancer involved, but in all cases the value has so far been significantly higher than in cancer-free individuals. The lowest IC5Q value to date for a cancer patient has been 270 vig/ml. However, an IC,-_ value above approx. 200, however, a high probability of. that the patient has a malignant neoplasm of one type or another.
Fremgangsmåten ifølge foreliggende oppfinnelse har med hell vært anvendt for påvisning av en lang rekke forskjellige krefttyper, og den antas derfor å være generelt anvendelig til kreftdiagnose. Som spesifikke eksempler-på påviste tumortyper . kan nevnes testikelcarcinoma, lymphoma, leukæmia, adenocarcinoma i brystet, småcellet adenocarcinoma i lungen, storcellet adenocarcinoma i lungen, adenocarcinoma av colon og melanoma, . for kun å nevne noen få. Fremgangsmåten synes også å være spesifikk til kreftdiagnose, idet kreft-frie pasienter med forskjellige typer non-neoplastiske sykdommer ikke har hatt de forhøyede IC^Q-verdier.som ledsager nærværet av kreft. The method according to the present invention has been successfully used for the detection of a large number of different types of cancer, and it is therefore assumed to be generally applicable for cancer diagnosis. As specific examples of proven tumor types. can be mentioned testicular carcinoma, lymphoma, leukaemia, adenocarcinoma of the breast, small cell adenocarcinoma of the lung, large cell adenocarcinoma of the lung, adenocarcinoma of the colon and melanoma, . to name just a few. The method also appears to be specific for cancer diagnosis, as cancer-free patients with various types of non-neoplastic diseases have not had the elevated IC^Q values that accompany the presence of cancer.
Den ovenfor beskrevne fluoressensprøve for inhibitor-protein X ifølge foreliggende oppfinnelse er basert på én ibo-ende egenskap hos dette stoff, nemlig inhiberingen av bleomycin-indusert nedbrytning av PM-2 DNA. Serumkonsentrasjonen av proteinet bestemt ved hjelp av denne fremgangsmåte har vært korre-lert med nærvær eller fraværet av malign sykdom.. Tilgjengelig-heten av et renset inhibitor-protein X muliggjør at en lang rekke innenfor biokjemien<p>g den kliniske kjemi kjente fremgangsmåte kan anvendes for dens måling. Fremtredende blant disse immunologiske metoder slik som radioimmunoundersøkelse, fluorimmunoundersøkeIse, enzymimmunoundersøkelse (f.eks. ELISA), spinimmunoundersøkelse, kjemiluminessens-immunoundersøkelse, fluoressens-polarisering-immunoundersøkelse, nephelometrisk undersøkelse, geldiffusjonsmetoder og klassiske metoder, slik som hemagglutinering og komplement-fixering. Videre kan andre funksjonelle egenskaper hos inhibitor-protein.X utnyttes til dets måling i biologiske væsker. Katalytiske fenomener i for-bindelse med molekylet kan måles ved spektrofotometriske eller spektrofluorometriske, kinetiske eller sluttpunkt prøver. Videre kan separeringsteknikk anvendes på den angjeldende væske og en lang rekke påvisningsmetoder kan anvendes til kvantise- . ring av proteinet, herunder spektrale eller densitometriske metoder og immunologiske metoder. Da det ved de foregående The above-described fluorescence test for inhibitor protein X according to the present invention is based on one inherent property of this substance, namely the inhibition of bleomycin-induced degradation of PM-2 DNA. The serum concentration of the protein determined using this method has been correlated with the presence or absence of malignant disease. The availability of a purified inhibitor protein X makes it possible that a wide range of methods known within biochemistry and clinical chemistry can used for its measurement. Prominent among these are immunological methods such as radioimmunoassay, fluoroimmunoassay, enzyme immunoassay (e.g. ELISA), spin immunoassay, chemiluminescence immunoassay, fluorescence polarization immunoassay, nephelometric assay, gel diffusion methods and classical methods, such as hemagglutination and complement fixation. Furthermore, other functional properties of inhibitor-protein.X can be exploited for its measurement in biological fluids. Catalytic phenomena in connection with the molecule can be measured by spectrophotometric or spectrofluorometric, kinetic or endpoint tests. Furthermore, separation techniques can be applied to the liquid in question and a wide range of detection methods can be used for quantification. ring of the protein, including spectral or densitometric methods and immunological methods. Then it by the previous ones
metoder oppnås spesifikk måling av konsentrasjonen av inhibitor-protein X, følger den at serumkonsentrasjonen av proteinet,, bestemt ved hjelp av slike fremgangsmåter, korrelerer med forekomsten eller fraværet av malign sykdom. methods specific measurement of the concentration of inhibitor protein X is obtained, it follows that the serum concentration of the protein, determined by such methods, correlates with the presence or absence of malignant disease.
Inhibitor-protein X renses i henhold til følgende metoder. Inhibitor protein X is purified according to the following methods.
Partiell rensing - Metode APartial purification - Method A
Inhibitor-protein X renses partielt ved "Sephadex" søy-iekromatografi. 1 ml humant serum settes til en "Sephadex G-75" kolonne på 88 cm x 2 cm og ble deretter eluert i en natriumfosfatpuffer på 0,07M og Ph 6,8 ved en strømningshastighet på 6-8 ml pr. time ved romtemperatur. Inhibitoren som var inneholdt i uttømningsvolumet som ble oppsamlet ble lyofilisert resuspendert i 1 ml 1^0, dialysert mot 20 volumdele H2O i 24 timer ved .4 C og deretter ført til en "Sephadex G-2 00" kolonne, som var ekvilibrert i samme puffer. Inhibitor protein X is partially purified by "Sephadex" column chromatography. 1 ml of human serum is added to a "Sephadex G-75" column of 88 cm x 2 cm and was then eluted in a sodium phosphate buffer of 0.07 M and Ph 6.8 at a flow rate of 6-8 ml per minute. hour at room temperature. The inhibitor contained in the depletion volume collected was lyophilized resuspended in 1 ml of 1^0, dialyzed against 20 volumes of H2O for 24 hours at .4 C and then applied to a "Sephadex G-2 00" column, which was equilibrated in the same puffer.
Eluering av "Sephadex G-200" kolonnen resulterte i tre. toppunkter som absorberte ved 280 nm. Toppunkt III som utgjør:; 7,7% av det totalt eluerte protein.inneholdt inhibitor-protein X. Dette ble behandlet som beskrevet ovenfor.og igjen satt, til en "Sephadex G-200" kolonne. Elueringen ble foretatt med den ovenfor beskrevne fosfatpuffer. 3 ml fraksjoner fra hver kolonne ble oppsamlet med en fraksjonskollektor (Gilson Medical Electronics, Inc., Middleton, Wis.) og elueringsmønstrene ble oppnådd ved absorbsjon ved 280 nm med et spektrofotometer. Proteinkonsentrasjoner ble bestemt ved hjelp av Lowry-metoden. Elution of the "Sephadex G-200" column resulted in three. peaks that absorbed at 280 nm. Vertex III which constitutes:; 7.7% of the total eluted protein contained inhibitor protein X. This was treated as described above and added again to a "Sephadex G-200" column. The elution was carried out with the phosphate buffer described above. 3 ml fractions from each column were collected with a fraction collector (Gilson Medical Electronics, Inc., Middleton, Wis.) and the elution patterns were obtained by absorbance at 280 nm with a spectrophotometer. Protein concentrations were determined using the Lowry method.
Rensing - Metode BCleaning - Method B
Rensing av inhibitor-proteinet ble oppnådd ved moleky-.. lær filtrering med "Amicbn" filtre, dialyse og "Sephadex" gel-søylekromatografi. De humane sera, 1-5 ml, ble sentrifugert i "Amicon Conical" filterrør (Amicon, Lexington, Mass.) ved 5000 Purification of the inhibitor protein was achieved by molecular filtration with "Amicbn" filters, dialysis and "Sephadex" gel column chromatography. The human sera, 1-5 ml, were centrifuged in "Amicon Conical" filter tubes (Amicon, Lexington, Mass.) at 5000
g i 10 minutter ved 4°C. Retentatet som ble tilbake i filter-kjernen etter sentrifugeringen ble resuspendert i deionisert vann til det opprinnelige serumvolum. Porsjoner av retentatet og filtratet ble prøvet på inhibitorisk aktivitet. Det aktive retentat ble dialysert i celluloseslanger mot 20 volumdeler deionisert vann ved 4°C i.24 timer. Retentatet og dialysatet ble lyofilisert og resuspendert i deionisert vann. Resultatene g for 10 minutes at 4°C. The retentate that remained in the filter core after centrifugation was resuspended in deionized water to the original serum volume. Portions of the retentate and filtrate were tested for inhibitory activity. The active retentate was dialyzed in cellulose tubes against 20 parts by volume of deionized water at 4°C for 24 hours. The retentate and dialysate were lyophilized and resuspended in deionized water. The results
av undersøkelsene av de to fraksjoner viste at den inhibitoriske aktivitet var i retentatet. Retentatet, 1 ml, ble satt til en 60 x 5 cm kolonne av "Sephadex G-50" og eluert med en natriumfosfatpuffer (0,07M, pH 6,8) ved en strømningshastighet på 30 . ml pr. time ved 4°C. Søyleeluatet>5 ml pr. glass, ble. samlet med en fraksjonskollektor av typen "Buchler Fractomette Alpha 200" (Searle, Fort Lee, N.J.). 280 nm absorbsjonen for hver fraksjon ble målt med et spektrofotpmeter og glassene blandes i henhold til absorbsjonstoppunktene. De blandede fraksjoner ble lyofilisert, dialysert og prøvet på inhibitorisk aktivitet som beskrevet ovenfor. Det "aktive materiale" som var i ut-tømningsvolumfraksjonen ble ført til "Sephadex G-75 og G-100" kolonner og eluert på samme måte som "G-50" kolonnen. Den inhibitoriske aktivitet ble funnet i uttømningsvolumfraksjonene for begge kolonner. Det "aktive" materiale som ble oppsamlet i "Sephadex G-200" kolonnens uttømningsvolumfraksjon ble eluert med natriumfosfatpuffer (0,07M, pH 6,8) ved romtemperatur ved en strømningshastighet på 6-8 ml pr. time. Fraksjonene ble samlet, 3 ml pr. glass, med en mikrofraksjonskollektor av typen "Gilson" (Gilsdn, Middleton, Wis,). Søyleeluatet ble fraksjo-nért på basis av 280 nm absorbsjons-profilen med sammenblanding av glassene for hvert av absorbsjons-toppunkt. Hver fraksjon i ble deretter lyofilisert, dialysert og prøvet på inhibitorisk aktivitet som beskrevet ovenfor. Den inhibitoriske aktivitet ble funnet i fraksjon III. Materialet i fraksjon III ble igjen satt til "Sephadex G-200" kolonnen og eluert under de samme betingelser. Fraksjon III fra "Sephadex G-200" kolonnen og som inneholdt inhibitor-protein X, representerte en rensing på mer enn 1000 ganger. of the investigations of the two fractions showed that the inhibitory activity was in the retentate. The retentate, 1 ml, was added to a 60 x 5 cm column of "Sephadex G-50" and eluted with a sodium phosphate buffer (0.07 M, pH 6.8) at a flow rate of 30 . ml per hour at 4°C. The column eluate>5 ml per glass, became collected with a "Buchler Fractomette Alpha 200" fraction collector (Searle, Fort Lee, N.J.). The 280 nm absorbance for each fraction was measured with a spectrophotometer and the glasses are mixed according to the absorbance peaks. The mixed fractions were lyophilized, dialyzed and tested for inhibitory activity as described above. The "active material" which was in the discharge volume fraction was fed to "Sephadex G-75 and G-100" columns and eluted in the same way as the "G-50" column. The inhibitory activity was found in the depletion volume fractions for both columns. The "active" material collected in the "Sephadex G-200" column discharge volume fraction was eluted with sodium phosphate buffer (0.07M, pH 6.8) at room temperature at a flow rate of 6-8 ml per minute. hour. The fractions were collected, 3 ml per glass, with a "Gilson" type microfraction collector (Gilsdn, Middleton, Wis,). The column eluate was fractionated on the basis of the 280 nm absorption profile with mixing of the glasses for each absorption peak. Each fraction i was then lyophilized, dialyzed and tested for inhibitory activity as described above. The inhibitory activity was found in fraction III. The material in fraction III was again added to the "Sephadex G-200" column and eluted under the same conditions. Fraction III from the "Sephadex G-200" column and containing inhibitor protein X represented a purification of more than 1000 times.
PM-2 DNA fluoressens-undersøkelsesmetoden for inhibitor-protein X er nærmere beskrevet nedenfor: The PM-2 DNA fluorescence examination method for inhibitor protein X is described in more detail below:
Bestemmelse av IC^ for inhibitor-protein XDetermination of IC^ for inhibitor protein X
Bestemmelsen av IC,.Q-verdien (den proteinkonsentrasjon som kreves for å gi 50% inhibering av PM-2 DNA nedbrytning) for inhibitor-protein X i humant serum foregår i to trinn: 1. Proteinkonsentrasjonen i serumet bestemmes ved Lowry-metoden. 2. PM-2 DNA fluoressensprøven for bleomycin anvendes for oppnåelse av en dosis-reåksjonskurve for serumproteininhibitor-ren. IC,-Q-verdiene avledes fra diagrammer av prosentual inhibering av DNA nedbrytning mot proteinkonsentrasjonene på onlog-probit grafer. Prosedyrer; The determination of the IC,.Q value (the protein concentration required to give 50% inhibition of PM-2 DNA degradation) for inhibitor protein X in human serum takes place in two steps: 1. The protein concentration in the serum is determined by the Lowry method. 2. The PM-2 DNA fluorescence sample for bleomycin is used to obtain a dose-response curve for the serum protein inhibitor. The IC,-Q values are derived from plots of percentage inhibition of DNA degradation against the protein concentrations on onlog-probit graphs. Procedures;
1. Lowry-reaksjonen1. The Lowry reaction
A. Fremstilling av basisoppløsninger:A. Preparation of base solutions:
Oppløsning A. 2% Na2C03 i 0,IN NaOHSolution A. 2% Na2C03 in 0.1N NaOH
Oppløsning B. 1% CuS04, 5H20 i H20Solution B. 1% CuS04, 5H2O in H2O
Oppløsning C. 2% Na<+>K<+>tartrat i H20Solution C. 2% Na<+>K<+>tartrate in H2O
Oppløsning D. 2N folin-ciocalteau-fenolreagensSolution D. 2N folin-ciocalteau-phenol reagent
B. Fremstilling av reagensoppløsninger:B. Preparation of reagent solutions:
Reagensoppløsning 1. 1 volum med basisoppløsning B Reagent solution 1. 1 volume of base solution B
blandes med 1 volum basisopp-løsning C. mixed with 1 volume of base solution C.
Reagensoppløsning 2. 50 volumer basisoppløsning A Reagent solution 2. 50 volumes of base solution A
blandes med 1 volum reagens-oppløsning 1. mixed with 1 volume of reagent solution 1.
Reagensoppløsning 3.;. 1 volum basisoppløsning D Reagent solution 3.;. 1 volume of base solution D
blandes med 1 volum H20.mixed with 1 volume of H20.
C. Reaksjoner:C. Reactions:
1. Serum fortynnes 1:10 med H20.1. Serum is diluted 1:10 with H20.
2. 10 yl fortynnet serum overføres til et 13 x 100 mm reagensglass og det tilsettes 290 yl H20. 3. 1 ml reagensoppløsning 2 tilsettes, det hele blandes grundig og inkuberes 10 minutter ved romtemperatur. 4. 100 yl av reagensoppløsning 3 tilsettes, det hélé blandes grundig og inkuberes 30 minutter ved romtemperatur . 5. Et spektrofotometer anvendes til måling av absorbsjons ved 750 nm. 6.. Proteinkonsentras jonen bestemmes utfra BSA standard-kurven. D. Okseserumalbumin (BSA) standardkurve: 2. 10 µl of diluted serum is transferred to a 13 x 100 mm test tube and 290 µl of H20 is added. 3. 1 ml of reagent solution 2 is added, the whole is mixed thoroughly and incubated for 10 minutes at room temperature. 4. Add 100 µl of reagent solution 3, mix thoroughly and incubate for 30 minutes at room temperature. 5. A spectrophotometer is used to measure absorbance at 750 nm. 6.. The protein concentration is determined from the BSA standard curve. D. Bovine serum albumin (BSA) standard curve:
1. Det fremstilles seriefortynninger av BSA, f.eks.1. Serial dilutions of BSA are prepared, e.g.
25, 50, 75, 100, 150 yg/ml, i et sluttvolum på 0,3 ml. 2. De i avsnitt D angitte reaksjoner foretas begyn-' nende ved trinn 3,. nemlig tilsetning av reagens-oppløsning 2. 3. Absorbsjon ved 750 nm føres opp mot BSA konsentrasjoner. 25, 50, 75, 100, 150 ug/ml, in a final volume of 0.3 ml. 2. The reactions specified in section D are carried out beginning with step 3. namely addition of reagent solution 2. 3. Absorbance at 750 nm is plotted against BSA concentrations.
II. ProteininhibitorundersøkelseII. Protein inhibitor assay
A. Det foretas seriefortynninger av serumproteinet på basis av de ved Lowry-reaksjonen bestemte serumproteinkonsen-trasjoner. Fortynninger fremstilles i et konstant volum natriumboratpuffer (0,05M, pH 9,5). A. Serial dilutions of the serum protein are made on the basis of the serum protein concentrations determined by the Lowry reaction. Dilutions are prepared in a constant volume of sodium borate buffer (0.05M, pH 9.5).
B. Prøver av serumprotein med volumer på 50 yl settes til 10 yl bleomycin (34,5 nanamol) inneholdt i et 13 x 100 mm reagensglass. C. Det tilsettes 390 yl natriumboratpuffer (0,05M, pH 9,5) inneholdende 25 mM 2-mercaptoetanol. D. Det tilsettes 50 yl PM-2 DNA (8,3 x 10-6M i 0,15M NaCl). B. Samples of serum protein in volumes of 50 µl are added to 10 µl of bleomycin (34.5 nanomoles) contained in a 13 x 100 mm test tube. C. 390 µl of sodium borate buffer (0.05 M, pH 9.5) containing 25 mM 2-mercaptoethanol is added. D. 50 µl PM-2 DNA (8.3 x 10-6M in 0.15M NaCl) is added.
E. Det inkuberes 30 minutter, ved 37°C.E. It is incubated for 30 minutes, at 37°C.
F. Triplikatporsjoner på. 100 yl overføres til 13 x 100 mm i reagensglass inneholdende 0,9 ml Na^PO^ (0/09M, pH 12,1) inneholdende 0,01M EDTA og 0,01MNaCl.. Det hele blandes grundig. F. Triplicate portions of. 100 µl are transferred to 13 x 100 mm test tubes containing 0.9 ml Na^PO^ (0/09M, pH 12.1) containing 0.01M EDTA and 0.01MNaCl.. The whole is mixed thoroughly.
G. Det tilsettes 100 yl ethidiumbromid (55,7 x 10-6M) i G. 100 μl of ethidium bromide (55.7 x 10-6M) is added in
Na3P04puffer, pH 12,1. Det hele blandes grundig.Na 3 PO 4 buffer, pH 12.1. The whole thing is thoroughly mixed.
H.. Kontrollreagenser:H.. Control reagents:
1.. Natriumborat (0,5M, pH 9,5) inneholdende 2-mercaptoetanol uten bleomycin eller proteiner. 1.. Sodium borate (0.5M, pH 9.5) containing 2-mercaptoethanol without bleomycin or proteins.
2. Protein uten bleomycin eller PM-2 DNA.2. Protein without bleomycin or PM-2 DNA.
3. Protein uten bleomycin.3. Protein without bleomycin.
4. Natriumborat (0,05M, pH 9,5) inneholdende 2-mercaptoetanol blindprøve. I. Spektrofotofluormetri (Aminco-Bowman): Prøven overføres til 1 cm kvartskuvette og fluoressensen måles ved 530 nm inngang og 590 emisjon. 4. Sodium borate (0.05M, pH 9.5) containing 2-mercaptoethanol blank. I. Spectrophotofluorometry (Aminco-Bowman): The sample is transferred to a 1 cm quartz cuvette and the fluorescence is measured at 530 nm input and 590 emission.
Claims (3)
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
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US2873279A | 1979-04-10 | 1979-04-10 |
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NO801018L true NO801018L (en) | 1980-10-13 |
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Application Number | Title | Priority Date | Filing Date |
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NO801018A NO801018L (en) | 1979-04-10 | 1980-04-09 | DIAGNOSTIC PROCEDURES. |
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JP (1) | JPS55143441A (en) |
KR (1) | KR830002502A (en) |
AR (1) | AR222700A1 (en) |
AT (1) | AT365343B (en) |
AU (1) | AU536522B2 (en) |
BE (1) | BE882669A (en) |
CH (1) | CH644453A5 (en) |
DE (1) | DE3013837A1 (en) |
DK (1) | DK149980A (en) |
ES (1) | ES8103380A1 (en) |
FI (1) | FI801078A (en) |
FR (1) | FR2454099A1 (en) |
GB (1) | GB2047712B (en) |
GR (1) | GR67692B (en) |
HU (1) | HU183122B (en) |
IE (1) | IE49981B1 (en) |
IL (1) | IL59772A (en) |
IT (1) | IT1143135B (en) |
LU (1) | LU82345A1 (en) |
NL (1) | NL8002094A (en) |
NO (1) | NO801018L (en) |
PH (1) | PH15637A (en) |
SE (1) | SE8002602L (en) |
ZA (1) | ZA802141B (en) |
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Publication number | Priority date | Publication date | Assignee | Title |
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DE3524644A1 (en) * | 1985-07-10 | 1987-01-15 | Heyl Chem Pharm | UNDULIN AND ITS PEPTIDE FRAGMENTS, METHOD FOR THE PRODUCTION THEREOF AND THEIR USE |
-
1980
- 1980-03-28 PH PH23825A patent/PH15637A/en unknown
- 1980-04-03 SE SE8002602A patent/SE8002602L/en not_active Application Discontinuation
- 1980-04-03 AU AU57193/80A patent/AU536522B2/en not_active Ceased
- 1980-04-03 IE IE693/80A patent/IE49981B1/en unknown
- 1980-04-03 FI FI801078A patent/FI801078A/en not_active Application Discontinuation
- 1980-04-04 BE BE0/200139A patent/BE882669A/en not_active IP Right Cessation
- 1980-04-04 IL IL59772A patent/IL59772A/en unknown
- 1980-04-04 FR FR8007691A patent/FR2454099A1/en active Granted
- 1980-04-07 JP JP4475080A patent/JPS55143441A/en active Pending
- 1980-04-08 DK DK149980A patent/DK149980A/en not_active IP Right Cessation
- 1980-04-08 LU LU82345A patent/LU82345A1/en unknown
- 1980-04-09 NO NO801018A patent/NO801018L/en unknown
- 1980-04-09 NL NL8002094A patent/NL8002094A/en not_active Application Discontinuation
- 1980-04-09 ES ES490396A patent/ES8103380A1/en not_active Expired
- 1980-04-09 IT IT48370/80A patent/IT1143135B/en active
- 1980-04-10 CH CH277880A patent/CH644453A5/en not_active IP Right Cessation
- 1980-04-10 HU HU80871A patent/HU183122B/en unknown
- 1980-04-10 AR AR280624A patent/AR222700A1/en active
- 1980-04-10 DE DE19803013837 patent/DE3013837A1/en not_active Withdrawn
- 1980-04-10 AT AT0195780A patent/AT365343B/en not_active IP Right Cessation
- 1980-04-10 ZA ZA00802141A patent/ZA802141B/en unknown
- 1980-04-10 GR GR61652A patent/GR67692B/el unknown
- 1980-04-10 KR KR1019800001518A patent/KR830002502A/en unknown
- 1980-04-10 GB GB8011902A patent/GB2047712B/en not_active Expired
Also Published As
Publication number | Publication date |
---|---|
FR2454099B1 (en) | 1984-01-27 |
IT1143135B (en) | 1986-10-22 |
HU183122B (en) | 1984-04-28 |
FR2454099A1 (en) | 1980-11-07 |
JPS55143441A (en) | 1980-11-08 |
IE800693L (en) | 1980-10-10 |
KR830002502A (en) | 1983-05-30 |
AR222700A1 (en) | 1981-06-15 |
AU5719380A (en) | 1980-10-16 |
ZA802141B (en) | 1981-04-29 |
IL59772A (en) | 1983-05-15 |
ATA195780A (en) | 1981-05-15 |
NL8002094A (en) | 1980-10-14 |
AU536522B2 (en) | 1984-05-10 |
GB2047712B (en) | 1983-02-23 |
ES490396A0 (en) | 1981-02-16 |
BE882669A (en) | 1980-10-06 |
GB2047712A (en) | 1980-12-03 |
PH15637A (en) | 1983-03-11 |
CH644453A5 (en) | 1984-07-31 |
FI801078A (en) | 1980-10-11 |
LU82345A1 (en) | 1980-12-16 |
GR67692B (en) | 1981-09-07 |
AT365343B (en) | 1982-01-11 |
DE3013837A1 (en) | 1980-10-30 |
DK149980A (en) | 1980-10-11 |
IE49981B1 (en) | 1986-01-22 |
IL59772A0 (en) | 1980-06-30 |
SE8002602L (en) | 1980-12-09 |
IT8048370A0 (en) | 1980-04-09 |
ES8103380A1 (en) | 1981-02-16 |
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