WO1990010872A1 - Gewinnung und verwendung von rna-proteolipidkomplex aus humanen malignen zellen - Google Patents

Gewinnung und verwendung von rna-proteolipidkomplex aus humanen malignen zellen Download PDF

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WO1990010872A1
WO1990010872A1 PCT/CH1990/000063 CH9000063W WO9010872A1 WO 1990010872 A1 WO1990010872 A1 WO 1990010872A1 CH 9000063 W CH9000063 W CH 9000063W WO 9010872 A1 WO9010872 A1 WO 9010872A1
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rna
proteolipid
cells
cdna
immunologically equivalent
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PCT/CH1990/000063
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German (de)
English (en)
French (fr)
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Kaspar Rhyner
Andreas Wieczorek
Peter Groscurth
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Kaspar Rhyner
Andreas Wieczorek
Peter Groscurth
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Publication of WO1990010872A1 publication Critical patent/WO1990010872A1/de

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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
    • 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
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • RNA-proteo-lipid complex Extraction and use of RNA-proteo-lipid complex from human malignant cells.
  • the invention relates to methods according to the preamble of claim 1 for the production of purified RNA proteolipid formed by cells of human neoplasms or their descendants and / or an immunologically equivalent lipophilic peptide fraction thereof and a method for the production of a labeled derivative thereof, methods for obtaining a monoclonal antibody against an RNA proteolipid formed by human malignant cells and / or against an immunologically equivalent lipophilic peptide fraction thereof and a method for obtaining a labeled derivative thereof, a method for obtaining one of human malignant cells formed RNA proteolipid of complementary deoxyribonucleic acid (cDNA), a method for obtaining single-stranded cDNA, and the substances obtained from these methods and uses thereof.
  • cDNA complementary deoxyribonucleic acid
  • a method according to the preamble of claim 1 for obtaining the RNA pro eolipid and / or an immunologically equivalent lipophilic peptide fraction thereof from sera from patients with neoplastic diseases and from cell culture supernatants of malignant cell lines has been described by A. Wieczorek, C. Rhyner and L. Block in "Proc. Natl. Acad. Sei. USA” 82: 3455-3459 (5/1985) and by A. Wieczorek, V. Sitaramam, W. Machleidt, K. Rhyner, A. Perruchoud and L. Block in Cancer Research 47: 6407-6412 (Dec. 1, 1987).
  • the technique for obtaining large quantities of a monoclonal antibody is generally known, but one that is Not yet known for the detection of human malignant cells or for the production of such agents useful monoclonal antibodies against said RNA proteolipid and / or the immunologically equivalent lipophilic peptide fractions thereof.
  • the object of the invention is to provide, with the help of the RNA proteolipid mentioned, an immunologically equivalent lipophilic peptide fraction thereof, an antibody, its labeled derivatives or a complementary cDNA agent which can be used to a large extent for diagnostic purposes and in particular one enable usable test to diagnose cancer.
  • RNA proteolipid formed by cells from Hu an neoplasia or their descendants and / or an immunologically equivalent lipophilic peptide fraction thereof.
  • a body fluid from neoplastic patients preferably blood serum, pleural or peritoneal fluid, or an extract from cultured human malignant cells of epithelial or mesenchymal origin, or a supernatant from a culture of such cells, becomes at least one potassium subjected to bromide density gradient ultracentrifugation.
  • the density range is about 1.080, which is on the boundary between the density ranges for ct lipoproteins (HDL) and 0 lipoproteins (LDL) and is recognizable below 11 ° C as an opalescent band at a to obtain the suspension of the RNA proteolipid and / or an immunologically equivalent lipophilic peptide fraction thereof.
  • body fluid that of those patients whose serum does not contain any immunochemically detectable lipoprotein a is used according to the invention, and the suspension obtained is subjected to decontamination by column chromatography over agarose according to the invention in order to separate it from ⁇ -lipoproteins (HDL). to free.
  • the decontaminated suspension is preferably further cleaned, in which is passed over an affinity column which has been provided by coupling anti-Hu serum globulins to agarose activated with cyanogen bromide with the purpose of removing further portions of at least lipoproteins and serum albumin therefrom.
  • the RNA proteolipid and / or an immunologically equivalent lipophilic peptide fraction thereof can be used according to the invention as an agent or for the preparation of agents for the detection of antibodies formed in living organisms against the RNA proteolipid or an immunologically equivalent lipophilic peptide fraction thereof.
  • a labeled derivative of the RNA proteolipid and / or an immunologically equivalent lipophilic peptide fraction thereof can also be provided by covalently binding the RNA proteolipid or an immunologically equivalent lipophilic peptide fraction thereof to a ligand.
  • the ligand is preferably a chelator, an enzyme, a radioactive compound, a fluorescent compound or a luminescent compound.
  • the labeled derivative can be used according to the invention as an agent and / or for the preparation of agents for the detection of antibodies formed in living organisms against the RNA proteolipid or an immunologically equivalent lipophilic peptide fraction thereof.
  • the object is achieved by the provision of a monoclonal antibody against an RNA proteolipid formed by cells of human neoplasms or their descendants and / or against immunologically equivalent lipophilic peptide fractions thereof.
  • clones of such hybridoma cells are used which produce a monoclonal antibody against the RNA proteolipid or an immunologically equivalent lipophilic peptide fraction thereof and which do not produce an antibody which reacts with normal serum, it being possible to allow the hybridoma cells to multiply in vivo.
  • the monoclonal antibody can be used according to the invention as an agent and / or for the preparation of agents for the detection of the RNA proteolipid formed by human malignant cells or an immunologically equivalent lipophilic Peptide fraction thereof.
  • a labeled derivative of the monoclonal antibody can also be provided by covalently binding it to a ligand.
  • the ligand is preferably a chelator, an enzyme, a radioactive compound, a fluorescent compound or a luminescent compound.
  • the marked derivative can be used as an agent and / or for the preparation of agents for the detection of the RNA proteolipid formed by human malignant cells or an immunologically equivalent lipophilic peptide fraction thereof.
  • RNA proteolipid of complementary deoxyribonucleic acid formed by human malignant cells.
  • This cDNA is obtained according to the invention by forming such cDNA which is complementary to the RNA from the RNA proteolipid, brings this cDNA into a bacteriophage, transforms Escherichia coli with the bacteriophage, clones and isolates the cDNA.
  • This cDNA is preferably converted into single-stranded cDNA.
  • this single-stranded cDNA can be used as an agent and / or for the preparation of agents for the detection of RNA proteolipids formed from human malignant cells or an immunologically equivalent lipophilic peptide fraction thereof.
  • RNA proteolipid and / or the immunologically equivalent lipophilic peptide fractions thereof represent a macromolecular complex which is composed of high molecular weight ribonucleic acid, cholesterol, phospholipids, lipid-bound sugars and lipophilic oligopeptides with a molecular weight of about 1000 to about 2200.
  • this complex ie in particular the RNA proteolipid and / or the immunologically equivalent lipophilic peptide fractions thereof, hereinafter subsumed under the name "RNA proteolipid complex”.
  • RNA-proteolipid complex It is assumed that the origin of the RNA-proteolipid complex lies in the malignant cells. This is based on tests with malignant cells established in vitro. oils which secrete the proteolipid " into the culture supernatant. Experimental studies on nude mice to which various human tumors have been implanted as xenografts have shown that the RNA-proteolipid complex can already be detected in very small tumors in serum Formation of the RNA-proteolipid complex appears to be mainly dependent on the malignant transformation of the cells, since the RNA-proteolipid complex is not detectable in animals with normal human implants such as fetal lung fragments, although these transplants grow in the recipient animals.
  • the concentration of the RNA-proteolipid complex in the serum increases with the growth of the tumor. After surgical removal of the tumor, the concentration of the RNA-proteolipid complex goes back to non-measurable values with a half-life of two days. Irradiation or chemotherapy also lead to a reduction in the RNA-proteolipid complex associated with the decrease in tumor mass. Upon the occurrence of relapses after first formerly ⁇ rich 'therapy it comes to a re-increase of RNA proteolipid complex in the serum. Accordingly, the RNA-proteolipid complex is suitable as a progression parameter for malignant tumors. In addition, the RNA-proteolipid complex also appears to be useful as a clinical parameter for malignant diseases.
  • RNA-proteolipid complex sought.
  • 102 patient sera were 30 malignant patients and 72 patients without malignant disease.
  • the malignant patients included the entire spectrum of malignant diseases, from acute leukemia to malignant lymphomas to all types of solid tumors such as bronchial carcinoma, breast carcinoma, teratocarcinoma, hypernephroma or prostate carcinoma. 28 of the 32 malignancies were identified by means of the test, which gives a sensitivity of 93%. 68 patients were correct negative, which corresponds to a specificity of 97%.
  • RNA-proteolipid complex can be obtained in a known manner (cf. Wieczorek et al., 1985, loc. Cit.) From the blood serum of patients with neoplastic diseases. However, only sera should be used that are not detected by immunochemical tests that are not marketed for lipoprotein a e cia.
  • RNA-proteolipid complex can also be obtained from supernatants from established malignant cell lines. Culture media conditioned by malignant human cells were examined. The cells grown from human tumors and established as permanent lines were obtained from Flow (Glasgow), Gibco (Bethesda) and Seromed (Munich): HEp-2, laryngeal carcinoma; KB, floor carcinoma; EB-2, Burkitt's lymphoma; HeLa, cervical cancer; Jlll, Monozytenleukal ie, Ht-1080, Fibrosarcoma; RD, rhabdomyosarcoma; without code, malignant melarioma of the skin. The following controls were used: skin fibroblasts from two healthy adults, lymphocytes and monocytes from a healthy adult, and fetal human lung cells and liver cells from Flow (Glasgow).
  • the cells growing in the monolayer were enriched in Eagle's Minimal Essential Medium, enriched with 1% non-essential amino acids and 10% - fetal bovine serum (Gibco) incubated.
  • Individual tests were carried out in the serum-free medium from Neuman and Tytell (Gibco) with and without the addition of LDL and HDL (100 .ug / ml) in order to test the influence of lipoproteins on the composition of the RNA-proteolipid complex.
  • HDL high-density lipoprotein 2
  • LDL low-density lipoprotein
  • VLDL, LDL and the opalescent band were sucked off successively from above.
  • the HDL bands and the lipoprotein-free shelter were also collected.
  • the equivalent fraction of the gradient was aspirated in serum samples which showed no opalescent band.
  • the opalescent fraction was most clearly visible using a metallic background.
  • the opalescence phenomenon is temperature-dependent. After the tubes are warmed up to room temperature, the visibility of the band decreases significantly. The samples were therefore viewed within 10 min and the bands marked. In the electron microscopic image, the particles (20-28 nm) appear larger than LDL (19-21 nm).
  • the opalescent band still contains contaminating serum proteins, which represent approximately 10% of the total amount. For this reason, at least a second ultracentrifugation is carried out on a potassium bromide density gradient, because in order to free the RNA-proteolipid complex in the opalescent band as well as possible from contaminating serum proteins.
  • RNA-proteolipid complex is passed over an affinity column, which is produced by coupling anti-human serum globulins to cyanogen bromide-activated agarose.
  • RNA-proteolipid complex as antigen was diluted in phosphate buffered saline (PBS) to a concentration of 150 .ug / ml peptide.
  • Rabbits were immunized three times with 50 ⁇ l of this solution at intervals of one week with the addition of complete Freund's adjuvant (1: 1). The injections were made on the inside of the thighs. The animals were bled after six weeks. The antibodies were cleaned (see Wieczorek et al., 1985, loc. Cit.).
  • BALB / C mice were injected three times with 0.5 ml each with 50 ⁇ g of the peptide fraction in PBS with the addition of . each 0.5 ml of Freund's complete adjuvant is immunized. The injections were carried out subcutaneously on days 0, 7 and 14. Another booster injection was performed on day 28.
  • the spleens of the animals were removed one week after the last injection in order to fuse spleen cells with mouse myeloma cells, using those myeloma cells which do not have the enzyme hypoxanthine guanine phosphoribosyl transferase (HGPRT) and therefore in a selective culture medium ( HAT medium) will not survive.
  • HGPRT hypoxanthine guanine phosphoribosyl transferase
  • the cell culture supernatants of the hybridoma cells were examined to determine whether they contain the desired monoclonal antibodies.
  • a dot blot ELISA was used.
  • RNA-proteolipid complex solution 10 ⁇ g / ml peptide
  • a second antibody diluted to 1: 10000 and labeled with peroxidase (Bio-Rad, goat antibody against mice in the globulin). After 1 h the mixture was washed again and developed with 0.1% 4-chloronaphtol in 20% methanol in PBS with 0.01% hydrogen peroxide.
  • AK33 IgM
  • AK37 IgG3
  • AK52 IgG3
  • the crude immunoglobulin fraction precipitated in this way was chromatographed with 0.1 M Tris-HCl (pH 8.2) over DEAE Affi-Gel Blue (Bio-Rad) according to the manufacturer's instructions, the immunoglobulin fractions were purified and concentrated using filters (Amicon XM50).
  • the Nakane, 1974 method was used. 10 mg of peroxidase (Serva, Heidelberg) were dissolved in 1.25 ml of distilled water and 250 ⁇ l of sodium periodate (43 mg / ml) were added. After stirring for 20 min at room temperature, 30 ⁇ l of ethylene glycol were added. After a further 10 min at room temperature, the sample was diluted to 10 ml with 1 mM sodium acetate (pH 4.4) and concentrated to 1 ml via filter (Amicon PM10). In parallel, 8 mg of antibody were dissolved in 1.18 ml of 0.2 M carbonate buffer (pH 9.0) and 0.82 ml of it was added to the peroxidase solution. After 2 h at room temperature the sample was cooled in an ice bath and 500 ⁇ l sodium borohydride in water (3.8 mg / ml) was added. The conjugate was over a Sephadex
  • RNA-proteolipid complex by ELISA
  • the sera were diluted with proportions 1:10, 1: 100, 1: 1000 and 1: 10000 with 0.5% sodium deoxycholate in PBS and 100 ⁇ l samples in polystyrene microtiter plates ( Linbro) pipetted. The incubation was carried out at 37 ° C because at the high deoxycholate concentration part of the sera gelled at room temperature. After 2 h, 20 ⁇ l of 3% bovine serum albumin in PBS were added to block.
  • the monoclonal antibodies were diluted to 10 ng / ml with PBS. Aliquots of 200 ul were pipetted into wells of microtiter plates. The incubation was carried out for 2 hours at room temperature. The mixture was then washed in succession with PBS and with 0.05% Tween 20 in PBS and incubated for 2 hours with 10% bovine serum albumin with the addition of 10% sucrose in PBS. After washing again with PBS, the plates were covered with parafilm and stored at 4 C until use. The plates coated with the capture antibody were incubated with diluted sera as in the direct ELISA. After 2 h at 37 ° C., the mixture was washed with PBS and 50 ⁇ l of the conjugated antibody were added. Incubation, washing and development were carried out as in the direct ELISA.
  • the plates coated with antibody were incubated for 2 hours with 5 ng RNA-proteolipid complex and 50 ⁇ l of the test serum diluted 1:10 with PBS.
  • the detection was carried out with antibody, with peroxidase or alkaline phosphatase had been labeled.
  • Antibody labeled with biotin was also used, in which case the biotin was detected with a streptavidin-peroxidase conjugate.
  • Ci / mol in the present case 5 / ug of purified peptide and 88 .ug of chloramine-T in 25 .ul of 50 mM sodium phosphate buffer (pH 7.5). After 1 min, 100 ⁇ l of a solution of 2.4 g / 1 sodium metabisulfite in the same buffer and then 200 ⁇ l of a solution of 10 g / 1 potassium iodide in the same buffer were added. The reaction mixture was applied to a 1 cm x 10 cm Sephadex 615 column and eluted with 50 mM of a phosphate buffer (pH 8.0) additionally containing 0.05% Tween 80.
  • a phosphate buffer pH 8.0
  • Polyvinyl plates (Costar) were coated with the antibody by means of ELISA as in the detection described above (20 ng immunoglobulin per well), and the plates were also blocked and stored as described there. 1 ng each of the radioactively labeled antigen as described above (approx. 10,000 cpm in 50 .ul) were incubated per well with 50 .ul of the serum diluted with PBS to 1:10 to 1: 1000. After 4 hours at room temperature, washing was carried out six times with PBS. The individual wells were cut out and counted in the scintillation process. The results were expressed in pg of the displaced 125I.
  • Serum aliquots of 3. ul were mixed with 3. ul 1% SDS in 0.1 N Tris acetate buffer (pH 8.0) and incubated at 37 ° C. for 6 h. Then the samples were separated on polyacrylamide gradient gels (2-16%, Pharmacia) (70 mA, 8 h). The gels were then covered with nitrocellulose and the separated molecules with the aid of 50 mM Tris-glycine buffer (pH 8.3) either electrophoretically (in the case of electro-immuno-blot) or by diffusion (in the case of western blot) onto the nitrocellulose transferred loose. The nitrocellulose sheets were blocked with 3% bovine serum albumin.
  • the leaves were incubated with the monoclonal antibodies in 1% bovine serum albumin for 2 h and after washing with PBS for 1 h in peroxidase-labeled antibodies against mouse IgG. After washing again, the color development was carried out with 0.1% 4-chloronaphtol in 20% methanol in PBS with 0.01% hydrogen peroxide.
  • RNA-proteolipid complex in cells and tissue circumcised by means of immunofluorescence, immunohistochemistry or immuno-gold labeling
  • RNA-proteolipid complex For the morphological detection of the RNA-proteolipid complex, unfixed cells or tissue sections (freeze, paraffin or plastic sections) pre-fixed with 2% paraformaldehyde were used, with various dilutions of the monoclonal antibodies listed above with 1% bovine albumin in PBS, incubated at room temperature. After an incubation of 12 h at 4 C, the samples were washed three times in PBS.
  • the preparations were then subjected to a second antibody (Bio-Rad, goat antibody to mouse immunoglobulins) (dilution 1:50) for 2 hours at room temperature, directed against mouse immunoglobulins and labeled with fluorescein isothiocyanate or with rhodamine treated.
  • the samples were then washed several times and, if they had not been pre-fixed, refixed with 2% formaldehyde in PBS.
  • the preparations covered with polyalcohol were examined in a fluorescence microscope provided with an incident fluorescence unit using specific filters.
  • the preparations were treated with a secondary antibody directed against mouse immunoglobulins and labeled with peroxidase (dilution 1:50) for 2 h at room temperature. After washing several times, the preparations were incubated with 0.02% hydrogen peroxide as substrate and with 3'-3-diaminobenzidine tetrahydrochloride or with 3-amino-9-ethyl-carbazole as chromogen.
  • antisera coupled with colloidal gold (5 nm) and directed against mouse immunoglobulins were used as secondary antibodies.
  • the gold marking is then strengthened by silver solutions (intense-LM, Jannssen).
  • the preparations were treated with PBS or with antibodies absorbed by the RNA-proteolipid complex instead of the primary antibody.
  • the mRNA from the complex was purified by removing the lipids and lipophilic peptides with chloroform / methanol, with 70% ethanol and finally with phenol-SDS and used for reverse transcription.
  • the mixture was incubated at 15 ° C. for 1 h. After a further incubation at 65 ° C. for 5 min, the samples were cooled on ice and centrifuged at 10,000 g for 2 s. The following batch was added to the supernatant: 400 .ul 30 mM sodium acetate (pH 4.5), 250 mM NaCl, 1 mM zinc sulfate, 5% glycerol, 20 ul Sul nuclease (1 U / ul, Sigma). The samples were incubated at 37 C for 1 h. Then 60 .ul 100 mM Tris / 100 mM EDTA were added.
  • M13 mp8 (brass, 1983, Amersha, host E. Coli JM 101) was used as the phage vector. These proven and easy-to-use phages proved to be suitable for producing large amounts of cDNA for hybridization tests.
  • the vector DNA was cut in parallel with the restriction endonucleases Bam HI and Pst I.
  • 50 ng of the vector in 1 ⁇ l of water were mixed with 10 ⁇ l of the reaction buffer (Bam HI: 10 mM Tris-HCl (pH 8.0), 100 mM NaCl, 10 mM MgC12, 1 mM mercaptoethanol, 100 ⁇ g / ml bovine serum albumin; Pst I: as above with 50 mM NaCl) and incubated for 3 h at 37 ° C.
  • the reaction buffer Bam HI: 10 mM Tris-HCl (pH 8.0), 100 mM NaCl, 10 mM MgC12, 1 mM mercaptoethanol, 100 ⁇ g / ml bovine serum albumin; Pst I: as above with 50 mM NaCl
  • the cDNA was linked to the endonucleases mentioned ligated.
  • the Bam HI linker 5'pd (CGGATCCG) and the Pst I linker 5'pd (GCTGCAGC) from Pharmacia were used.
  • the cDNA flanked with linkers was in the buffers mentioned for 30 min at 37 ° C. with the endonucleases Bam HI and Pst
  • 40 ⁇ g of the cut vector DNA and 40 ⁇ g of the flanked cDNA were dissolved in 5 ⁇ l of water, combined and the following mixture was added: 2 ⁇ l of 150 M Tris-HCl (pH 8.0), 20 mM MgC12 , 6 mM EDTA, 5 mM dithiothreitol, 10 mM ATP,
  • the host cells (Escherichia Coli JM 101) were streaked on glucose minimal medium agar, which per liter 15 g agar (Gibco), 6 g Na. 2 HP0 4 , 3 g H 2 P0 4 , 1 g NH ⁇ Cl, 0.5 g NaCl, 1 ml IM MgS0 4 , 1 ml 0.1 M CaCl 2 , 1 ml 1 M thiamine and 10 ml Contained 20% glucose.
  • This mixture was combined with the transformed cells (0.2 ml) and mixed with an agar solution kept at 42 ° C. which contained 10 g of Bakotrypton, 8 g of NaCl and 8 g of agar per liter. This solution was immediately poured onto agar plates (10 g of baktotrypton, 8 g of NaCl, 12 g of agar per liter).
  • plaques were observed. About 5% of them were blue, i.e. they contained the self-ligated vector, which contains no cDNA.
  • the colorless plaques were transferred with sterilized toothpicks to 2 ml of the above-mentioned liquid medium and incubated overnight. The method described is a modified embodiment of known methods (Messing, 1983, Gray et al. 1983).
  • the cultures of the individual plaques were centrifuged (5000 g, 5 min).
  • the supernatants (1.6 ml) were mixed with 0.3 ml of 20% polyethylene glycol (MG 6000-8000, Sigma) and incubated at 22 C for 20 min. After centrifugation (10,000 g, 5 min) the supernatant was discarded. Remains of Polyethylene glycol solution was removed with filter paper.
  • the phages were suspended in 100 ⁇ l of 10 M Tris-HCl / 1 mM EDTA. Then 50 ⁇ l phenol / chloroform / isoamyl alcohol solution (49.5: 49.5: 1) were added. The samples were shaken and centrifuged at 10,000 g for 5 min. The upper phases were transferred to 1.5 ml Eppendorf tubes and the extraction of the cDNA with diethyl ether and the precipitation of the recombinant DNA with ethanol were carried out as described above.
  • the DNA precipitates were dissolved in 100 mM Tris acetate / 1 mM EDTA and 10 ⁇ l aliquots in 1% Agaro ⁇ e gels were separated in the same buffer (40 mA, 30 min). The gels were incubated for 15 minutes in 1.ug / ml ethidium bromide and viewed in long-wave UV light (300 nm).
  • the DNA is dissolved in a concentration of 0.5 .ug / ml in distilled water, heated at 100 ° C. for 10 minutes and then immediately cooled in an ice bath. 50 .ul of solution A and 12.5 .ul of solution B are added to 100 .ul of this solution. After 12 h of incubation, 350 ⁇ l of ethanol were added. After cooling to -20oC over a, the DNA was centrifuged off (14000 g,
  • RNA-proteolipid complex from the sera was pre-purified by affinity absorption. 1 l samples of the undiluted sera were mixed with 250 .ul 3 M SSC and 150 .ul 1% Nonidet P-40. Pieces of polyurethane paper (0.5 x 1 cm, Hybond-mAP, Amersham) were placed in the tubes and the samples were incubated for 12 h. The RNA-proteoplipid complex is stabilized by the detergent so that the polyadenyl chain of the mRNA contained in the RNA-proteoplipid complex can bind to the paper-bound polyuridine chain.
  • the serum was pipetted off and the paper pieces were washed three times with 2 ml of 0.5 M NaCl, then with 2 ml of 70% ethanol and then with 2 ml of cold distilled water. Then 1.5 ml of hot water (80 ° C.) were added, the piece of paper was removed, the solution was cooled, 200 ⁇ l of 1 M sodium acetate and 5 ml of ethanol were added and the mixture was cooled to ⁇ 20 ° C.
  • the mRNA partially purified in this way was centrifuged off (14000 g, 20 min), dissolved in 50 ⁇ l of 50 mM Tris-HCl, heated to 90 ° C. for 5 min and then immediately cooled in an ice bath.
  • the monoclonal antibody against the sulfonated DNA (Sigma) was diluted 1: 300 in the blocking buffer. After 1 h, washing was carried out with 100 mM Tris-HCl, (pH 7.5), 0.5 M NaCl and then second antibody labeled with peroxidase (Bio-Rad, goat antibody against mouse immunoglobulins) 1: 500 in the bloc Kier buffer diluted. After a further 1 h, the mixture was washed again with 100 mM Tris-HCl (pH 7.5), 0.5 M NaCl and the membrane was immersed in the color developer solution (0.1% 4-chloro-naphthol in 4% strength) Methanol in PBS with 0.01% hydrogen peroxide). After 10 min the membrane was washed with PBS and dried. The intensity of the color spots was compared with that of color spots of purified and pre-quantified RNA-proteolipid complex.

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PCT/CH1990/000063 1989-03-15 1990-03-12 Gewinnung und verwendung von rna-proteolipidkomplex aus humanen malignen zellen WO1990010872A1 (de)

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1992002249A1 (de) * 1990-08-08 1992-02-20 Viktor Balazs VERFAHREN ZUR VERHINDERUNG DER FUNKTIONSAUSÜBUNG DER GESCHÜTZTEN EXTRAZELLULAREN mRNAs VON KREBSZELLURSPRUNG FÜR PROPHYLAXE BÖSARTIGER KRANKHEITEN BZW. DEREN RELAPSE
US5591625A (en) * 1993-11-24 1997-01-07 Case Western Reserve University Transduced mesenchymal stem cells
WO2010079118A1 (en) 2009-01-07 2010-07-15 Michael Roth-Chiarello Use of rna obtained from proteolipid complexes circulating in blood for diagnosis and treatment of tumors
US8476017B2 (en) 2004-06-02 2013-07-02 Proxy Life Sciene Holdings, Inc. Microvesicle-based compositions and methods

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WO1992002249A1 (de) * 1990-08-08 1992-02-20 Viktor Balazs VERFAHREN ZUR VERHINDERUNG DER FUNKTIONSAUSÜBUNG DER GESCHÜTZTEN EXTRAZELLULAREN mRNAs VON KREBSZELLURSPRUNG FÜR PROPHYLAXE BÖSARTIGER KRANKHEITEN BZW. DEREN RELAPSE
US5591625A (en) * 1993-11-24 1997-01-07 Case Western Reserve University Transduced mesenchymal stem cells
US8476017B2 (en) 2004-06-02 2013-07-02 Proxy Life Sciene Holdings, Inc. Microvesicle-based compositions and methods
WO2010079118A1 (en) 2009-01-07 2010-07-15 Michael Roth-Chiarello Use of rna obtained from proteolipid complexes circulating in blood for diagnosis and treatment of tumors

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