DK168872B1 - Method for producing antibodies for immunological determination of amino-terminal procollagen peptide (type III) and / or procollagen (type III), antibodies produced by the method, use of the antibodies for immunological determination of amino-terminal procollagen peptide (type III) and / or procollagen (type III) and starting product for use in the process - Google Patents
Method for producing antibodies for immunological determination of amino-terminal procollagen peptide (type III) and / or procollagen (type III), antibodies produced by the method, use of the antibodies for immunological determination of amino-terminal procollagen peptide (type III) and / or procollagen (type III) and starting product for use in the process Download PDFInfo
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- DK168872B1 DK168872B1 DK232988A DK232988A DK168872B1 DK 168872 B1 DK168872 B1 DK 168872B1 DK 232988 A DK232988 A DK 232988A DK 232988 A DK232988 A DK 232988A DK 168872 B1 DK168872 B1 DK 168872B1
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- 108090000765 processed proteins & peptides Proteins 0.000 title claims abstract description 46
- 108010050808 Procollagen Proteins 0.000 title claims abstract description 43
- 238000000034 method Methods 0.000 title claims description 23
- 230000001900 immune effect Effects 0.000 title claims description 9
- 238000004519 manufacturing process Methods 0.000 title claims description 4
- 102000004169 proteins and genes Human genes 0.000 claims abstract description 10
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 10
- 241001465754 Metazoa Species 0.000 claims abstract description 9
- 230000002163 immunogen Effects 0.000 claims abstract description 7
- 210000002966 serum Anatomy 0.000 claims description 11
- 241000283973 Oryctolagus cuniculus Species 0.000 claims description 5
- 108060003552 hemocyanin Proteins 0.000 claims description 4
- 102000009027 Albumins Human genes 0.000 claims description 3
- 108010088751 Albumins Proteins 0.000 claims description 3
- 241000283707 Capra Species 0.000 claims description 3
- 108010039918 Polylysine Proteins 0.000 claims description 3
- 241000283984 Rodentia Species 0.000 claims description 3
- 229920000656 polylysine Polymers 0.000 claims description 3
- 241001494479 Pecora Species 0.000 claims 2
- 210000001124 body fluid Anatomy 0.000 abstract description 8
- 239000010839 body fluid Substances 0.000 abstract description 8
- 102000001187 Collagen Type III Human genes 0.000 abstract description 2
- 108010069502 Collagen Type III Proteins 0.000 abstract description 2
- 239000000427 antigen Substances 0.000 description 11
- 102000036639 antigens Human genes 0.000 description 11
- 108091007433 antigens Proteins 0.000 description 11
- 238000010790 dilution Methods 0.000 description 5
- 239000012895 dilution Substances 0.000 description 5
- 239000000047 product Substances 0.000 description 4
- 239000007857 degradation product Substances 0.000 description 3
- 230000000694 effects Effects 0.000 description 3
- 230000003053 immunization Effects 0.000 description 3
- 238000002649 immunization Methods 0.000 description 3
- 238000002360 preparation method Methods 0.000 description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 3
- 238000003556 assay Methods 0.000 description 2
- 150000001875 compounds Chemical class 0.000 description 2
- LOKCTEFSRHRXRJ-UHFFFAOYSA-I dipotassium trisodium dihydrogen phosphate hydrogen phosphate dichloride Chemical compound P(=O)(O)(O)[O-].[K+].P(=O)(O)([O-])[O-].[Na+].[Na+].[Cl-].[K+].[Cl-].[Na+] LOKCTEFSRHRXRJ-UHFFFAOYSA-I 0.000 description 2
- 238000003018 immunoassay Methods 0.000 description 2
- 239000002953 phosphate buffered saline Substances 0.000 description 2
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 2
- 238000000159 protein binding assay Methods 0.000 description 2
- 238000003127 radioimmunoassay Methods 0.000 description 2
- 239000000243 solution Substances 0.000 description 2
- 238000003756 stirring Methods 0.000 description 2
- 239000006228 supernatant Substances 0.000 description 2
- KYRUKRFVOACELK-UHFFFAOYSA-N (2,5-dioxopyrrolidin-1-yl) 3-(4-hydroxyphenyl)propanoate Chemical compound C1=CC(O)=CC=C1CCC(=O)ON1C(=O)CCC1=O KYRUKRFVOACELK-UHFFFAOYSA-N 0.000 description 1
- 241000700198 Cavia Species 0.000 description 1
- 108010035532 Collagen Proteins 0.000 description 1
- 102000008186 Collagen Human genes 0.000 description 1
- RWSOTUBLDIXVET-UHFFFAOYSA-N Dihydrogen sulfide Chemical compound S RWSOTUBLDIXVET-UHFFFAOYSA-N 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 108010037362 Extracellular Matrix Proteins Proteins 0.000 description 1
- 102000010834 Extracellular Matrix Proteins Human genes 0.000 description 1
- 229920001213 Polysorbate 20 Polymers 0.000 description 1
- 239000012506 Sephacryl® Substances 0.000 description 1
- 241000282887 Suidae Species 0.000 description 1
- 239000002671 adjuvant Substances 0.000 description 1
- 125000003275 alpha amino acid group Chemical group 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 235000015278 beef Nutrition 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 239000012888 bovine serum Substances 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- 210000004899 c-terminal region Anatomy 0.000 description 1
- 230000015556 catabolic process Effects 0.000 description 1
- 238000012512 characterization method Methods 0.000 description 1
- 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
- 229920001436 collagen Polymers 0.000 description 1
- 238000012875 competitive assay Methods 0.000 description 1
- 238000003271 compound fluorescence assay Methods 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 238000009826 distribution Methods 0.000 description 1
- 238000010828 elution Methods 0.000 description 1
- 210000002744 extracellular matrix Anatomy 0.000 description 1
- 230000003176 fibrotic effect Effects 0.000 description 1
- 238000005194 fractionation Methods 0.000 description 1
- 238000004108 freeze drying Methods 0.000 description 1
- 238000001641 gel filtration chromatography Methods 0.000 description 1
- 230000028993 immune response Effects 0.000 description 1
- 229940027941 immunoglobulin g Drugs 0.000 description 1
- 238000010324 immunological assay Methods 0.000 description 1
- 230000005764 inhibitory process Effects 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- 238000002372 labelling Methods 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 210000004185 liver Anatomy 0.000 description 1
- MYWUZJCMWCOHBA-VIFPVBQESA-N methamphetamine Chemical compound CN[C@@H](C)CC1=CC=CC=C1 MYWUZJCMWCOHBA-VIFPVBQESA-N 0.000 description 1
- HQXKKQDTQDYERH-UHFFFAOYSA-N n'-cyclohexyl-n-(2-morpholin-4-ylethyl)methanediimine;methyl 4-methylbenzenesulfonate Chemical compound COS(=O)(=O)C1=CC=C(C)C=C1.C1CCCCC1N=C=NCCN1CCOCC1 HQXKKQDTQDYERH-UHFFFAOYSA-N 0.000 description 1
- 108010033916 pN-collagen type III Proteins 0.000 description 1
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 description 1
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 description 1
- 102000004196 processed proteins & peptides Human genes 0.000 description 1
- 238000012207 quantitative assay Methods 0.000 description 1
- 239000011541 reaction mixture Substances 0.000 description 1
- 230000028327 secretion Effects 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 210000001519 tissue 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]
-
- 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
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
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- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Organic Chemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Biophysics (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Biochemistry (AREA)
- Zoology (AREA)
- Toxicology (AREA)
- Immunology (AREA)
- Gastroenterology & Hepatology (AREA)
- Peptides Or Proteins (AREA)
- Investigating Or Analysing Biological Materials (AREA)
- Lifting Devices For Agricultural Implements (AREA)
- Tires In General (AREA)
- Agricultural Machines (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
Description
DK 168872 Bl iDK 168872 Bl i
Opfindelsen angår en fremgangsmåde til fremstilling af antistoffer til immunologisk bestemmelse åf aminoterminalt prokollagen peptid (type III) og/eller prokollagen (type III), antistoffer fremstillet ved fremgangsmåden, anvendelsen 5 af antistofferne til immunologisk bestemmelse af aminoterminalt prokollagen peptid (type III) og/eller prokollagen (type III) og udgangsprodukt til brug ved fremgangsmåden.The invention relates to a method for producing antibodies for immunological determination of amino-terminal procollagen peptide (type III) and / or procollagen (type III), antibodies produced by the method, the use of the antibodies for immunological determination of amino-terminal procollagen peptide (type III) and / or procollagen (type III) and starting product for use in the process.
Prokollagen peptid (type III) er det aminoter-minale propeptid af kollagen (type III), der efter secer-10 nering af prokollagen (type III)-molekyler bliver fraspaltet extracellulært. Ved hjælp af en radioimmunologisk bestemmelsesmetode, som den der beskrives i europæisk patentskrift nr. 4940, kan koncentrationen af dette prokollagen peptid bestemmes i legemsvæsker. Viden om peptidets 15 serumskoncentration tillader udsagn om de fibrotiske sygdommes aktivitet, f.eks. i leveren (Rohde, H. et al.,Procollagen peptide (type III) is the amino-terminal propeptide of collagen (type III) that is cleaved extracellularly upon secretion of procollagen (type III) molecules. Using a radioimmunological assay method, such as that described in European Patent No. 4940, the concentration of this procollagen peptide can be determined in body fluids. Knowledge of the peptide's serum concentration allows statements about the activity of fibrotic diseases, e.g. in the liver (Rohde, H. et al.,
Eur. J. Clin. Invest. 9, 451-459 (1979)).Eur. J. Clin. Invest. 9, 451-459 (1979)).
Præcis selektiv bestemmelse af prokollagen peptid (type III) i serum og andre legemsvæsker er dog ikke 20 mulig med de hidtil beskrevne metoder, da de polyklonale antistoffer, der anvendes i disse metoder, reagerer med divergerende lavere affinitet med forskellige i serum forekommmende antigener, der til dels er nedbrydningsprodukter af prokollagen peptid (type III) (Niemelå, 0. et 25 al., Clin. Chim. Acta 124, 39-44 (1982)). Dette fører til, at serumsfortyndingskurverne og fortyndingskurverne for andre legemsvæsker ikke er parallele med standardkurven, hvilken er fremstillet med rent prokollagen peptid (type III), og derfor skal antigenindholdet i flere fortyn-30 dinger bestemmes for hver ukendt prøve, for at måle antigenkoncentrationen via fortyndingskurvens 50 % intercept.However, precise selective determination of procollagen peptide (type III) in serum and other body fluids is not possible with the methods described so far, as the polyclonal antibodies used in these methods react with divergent lower affinity with various serum antigens which in part, degradation products of procollagen peptide (type III) (Niemelå, 0. et al., Clin. Chim. Acta 124, 39-44 (1982)). This means that the serum dilution curves and the dilution curves for other body fluids are not parallel to the standard curve produced with pure procollagen peptide (type III) and therefore the antigen content in multiple dilutions must be determined for each unknown sample to measure antigen concentration via dilution curve 50% intercept.
En yderligere ulempe ved disse fremgangsmåder er, at det på grund af en for. ringe krydsaktivitet mellem 35 antistoffet og rotte- eller museantigen ikke er muligt med en bestemmelse af antigenkoncentrationen i legemsvæsker 2 DK 168872 B1 fra disse arter.A further disadvantage of these methods is that due to a feed. little cross-activity between the antibody and rat or mouse antigen is not possible with a determination of the antigen concentration in body fluids from these species.
Ved hjælp af den fremgangsmåde, der er beskrevet af Schuppan et al. /j. Hepatol. , 27-37 (1986)7, kan koncentrationen af antigener i rotteserum bestemmes.Using the method described by Schuppan et al. / J. Hepatol. , 27-37 (1986) 7, the concentration of antigens in rat serum can be determined.
5 Ganske vist har denne fremgangsmåde den samme ulempe, som også er beskrevet ved metoden for humane sera /Tliemelå et al., Clin. Chim. Acta 124, 39-44 (1982)7 med de ikke parallele inhibitionskurver ved forskellige legemsvæsker.5 Although this method has the same disadvantage also described by the method of human sera / Tliemelå et al., Clin. Chim. Acta 124, 39-44 (1982) 7 with the non-parallel inhibition curves of different body fluids.
10 Ved hjælp af fremgangsmåden i europæisk patent ansøgning nr. 0089.008 er det muligt at løse dette tekniske problem, idet der anvendes antistoffer, der har sammenlignelig affinitet for intakt prokollagen peptid (type III) og nedbrydningsproduktet heraf Col 1. Ved 15 hjælp af denne fremgangsmåde kan intakt og nedbrudt prokollagen peptid (type III) bestemmes i fællesskab, hvilket dog fører til unøjagtighed ved det diagnostiske udsagn, da normalkollektiv og patientkollektiv, kan overlappe kraftigt.By the method of European Patent Application No. 0089,008, it is possible to solve this technical problem using antibodies having comparable affinity for intact procollagen peptide (type III) and its degradation product Col 1. By this method For example, intact and degraded procollagen peptide (type III) can be determined jointly, which, however, leads to inaccuracy in the diagnostic statement, as the normal and patient collective may overlap sharply.
20 Det har nu overraskende vist sig, at ved an vendelse af et peptid med en bestemt aminosyresekvens til immuniseringen kan der opnås antistoffer, ved hjælp af hvilke en specifik bestemmelse af det intakte prokollagen peptid (type III) er mulig. Ved hjælp af disse an-25 tistoffer kan indholdet af prokollagen peptid (type III) i legemsvæsker fra rotter eller mus overraskende også bestemmes, hvilket er gavnligt ved den dyreeksperimen-telle afprøvning af fibrosuppressive substanser.Surprisingly, it has now been found that using a peptide with a particular amino acid sequence for the immunization can be obtained antibodies by which a specific determination of the intact procollagen peptide (type III) is possible. Surprisingly, with the help of these antibodies, the content of procollagen peptide (type III) in rat or mouse body fluids can also be determined, which is beneficial in the animal experimental testing of fibrosuppressive substances.
Opfindelsen angår således en fremgangsmåde til 30 fremstilling af antistoffer til immunologisk bestemmelse af aminoterminalt prokollagen peptid (type III) og/eller prokollagen (type III), hvilken fremgangsmåde er ejendommelig ved, at a) dyr bliver immuniseret med et peptid med sekvensen 35 I-C—E-S-C-P-T-G-G-Q-N-Y-S-P- bundet til et immunogent protein, og 3 DK 168872 B1 b) der ud fra serumen udvindes antistoffer, der reagerer med intakt aminoterminalt prokollagen peptid (type III).Thus, the invention relates to a method for producing antibodies for immunologically determining amino-terminal procollagen peptide (type III) and / or procollagen (type III), characterized in that a) animals are immunized with a peptide having the sequence 35 IC- ESCPTGGQNYSP bound to an immunogenic protein, and b) antibodies derived from the intact amino-terminal procollagen peptide (type III) are recovered from the serum.
Opfindelsen angår også antistoffer, der er ejendommelige ved, at de kan fremstilles ved fremgangsmåden ifølge 5 opfindelsen.The invention also relates to antibodies which are characterized in that they can be prepared by the method of the invention.
Opfindelsen angår endvidere anvendelsen af disse antistoffer ved en immunologisk bestemmelse af aminoterminalt prokollagen peptid (type III) og/eller prokollagen (type III) .The invention further relates to the use of these antibodies in an immunological determination of amino-terminal procollagen peptide (type III) and / or procollagen (type III).
10 Opfindelsen angår endelig også et udgangsprodukt til brug ved fremgangsmåden ifølge opfindelsen, hvilket udgangsprodukt er ejendommeligt ved, at det består af et peptid med sekvensen I-C-E-S-C-P-T-G-G-Q-N-Y-S-P 15 der er bundet til et immunogent protein.Finally, the invention also relates to a starting product for use in the method of the invention, which is characterized in that it consists of a peptide having the sequence I-C-E-S-C-P-T-G-G-Q-N-Y-S-P-15 bound to an immunogenic protein.
I det følgende beskrives opfindelsen detaljeret, især i dens foretrukne udførelsesformer.In the following, the invention is described in detail, especially in its preferred embodiments.
Til fremstillingen af antistoffet immuniseres dyr, fortrinsvis gnavere, såsom kaniner eller marsvin,For the preparation of the antibody, animals, preferably rodents such as rabbits or guinea pigs, are immunized.
20 eller geder og får med peptidet med sekvensen I-C-E-S-C-P-T-G-G-Q-N-Y-S-P20 or goats and with the peptide having the sequence I-C-E-S-C-P-T-G-G-Q-N-Y-S-P
koblet til et egnet immunogent protein i nærværelse af en komplet adjuvans. Især foretrukket anvendes der kaniner. Med gentagne sekundære injektioner, f.eks. med et 25 mellemrum på 4 til 8 uger, forstærkes immunsvaret. Resultatet af immuniseringen kontrolleres ved bestemmelse af koncentrationen af antistoffer ved den radioimrounologiske bindingsundersøgelse /R. Timpl og L. Risteli, Immunoche-mistry of the extracellular matrix, H. Furthmayr Ed., 30 Vol. 1, 199 (1982)7.coupled to a suitable immunogenic protein in the presence of a complete adjuvant. In particular, rabbits are used. With repeated secondary injections, e.g. every 25 to 4 weeks, the immune response is enhanced. The result of immunization is checked by determining the concentration of antibodies in the radioimmunological binding assay / R. Timpl and L. Risteli, Immunochemistry of the Extracellular Matrix, H. Furthmayr Ed., 30 Vol. 1, 199 (1982) 7.
Egnede proteiner, til hvilke det nævnte peptid kan være bundet, er alle immunogene proteiner. Fortrinsvis anvendes hæmocyanin, albumin eller polylysin. Peptidet kan fremstilles efter af fagfolk kendte metoder, som 35 f.eks. beskrevet af G. Barany og A.B. Merrifield i TheSuitable proteins to which the said peptide may be bound are all immunogenic proteins. Preferably, hemocyanin, albumin or polylysine is used. The peptide can be prepared by methods known in the art, such as e.g. described by G. Barany and A.B. Merrifield in The
Peptides, Vol. 2, side 3-254 (1980), Academic Press eller DK 168872 B1 4Peptides, Vol. 2, page 3-254 (1980), Academic Press or DK 168872 B1 4
OISLAND
af E. Brown, R.C. Sheppard og B.J. Williams, J. Chem. Soc. Perkin Transact. , 1161 (1983).by E. Brown, R.C. Sheppard and B.J. Williams, J. Chem. Soc. Perkin Transact. , 1161 (1983).
Antistofferne ifølge opfindelsen kan anvendes som serum eller oprenset i forskellige immunologiske frem-5 gangsmåder, inklusiv alle former for radioimmunanalyse, f.eks. sekventiel mætningsanalyse eller ligevægtsanalyse, mærkning med chloramin T eller Bolton-Hunter-reagens /Felber, Meth. Biochem. Anal. 22, 1 (1974), Shelley et al., Clin. Chem. 1_£, 146 (1975)^7 samt i andre kompetiti-10 ve bindingsanalyser, såsom fluorescensanalyse, enzymimmu-noanalyse, kemiluminescens- eller andre immunoanalyser. Antistofferne kan derfor anvendes til immunologiske fremgangsmåder til isolering og karakteriséring samt til kvantitativ bestemmelse af prokollagen peptid (type III) i 15 væv og legemsvæsker. Til kvantitativ bestemmelse gås frem efter af fagfolk i og for sig kendte metoder, idet en flydende prøve, der indeholder prokollagen peptid (type III), bringes til reaktion med antistofferne ifølge opfindelsen, og mængden af prokollagen peptid (type III) bestemmes via 20 det dannede antigen-antistof-kompleks. Derved spiller det ingen rolle, om prokollagen peptidet (type III) endnu er sammenbundet med aminoterminalen på prokollagen (type III) eller ikke. De hidtil ved den immunologiske bestemmelse forstyrrende nedbrydningsprodukter af prokollagen pepti-25 det (type III), især Col 1, medtages ikke af antistofferne ifølge opfindelsen.The antibodies of the invention can be used as serum or purified in various immunological methods, including all forms of radioimmunoassay, e.g. sequential saturation or equilibrium analysis, labeling with chloramine T or Bolton-Hunter reagent / Felber, Meth. Biochem. Anal. 22, 1 (1974), Shelley et al., Clin. Chem. 1, 146 (1975) 7, and in other competitive binding assays such as fluorescence assay, enzyme immunoassay, chemiluminescence or other immunoassays. The antibodies can therefore be used for immunological methods for isolation and characterization as well as for the quantitative determination of procollagen peptide (type III) in 15 tissues and body fluids. For quantitative assay, methods are known in the art as a liquid sample containing procollagen peptide (type III) is reacted with the antibodies of the invention and the amount of procollagen peptide (type III) is determined via the formed antigen-antibody complex. Thus, it does not matter whether or not the procollagen peptide (type III) is linked to the amino terminus of procollagen (type III). The disruptive breakdown products of the procollagen peptide (type III), in particular Col 1, which are disturbed by the immunological assay, are not included in the antibodies of the invention.
I de følgende eksempler illustreres opfindelsen yderligere. Procentangivelser er baseret på vægten, såfremt andet ikke er anført.The following examples further illustrate the invention. Percentages are based on the weight, unless otherwise stated.
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Eksempel 1Example 1
Fremstilling af peptid-hæmocyanin konjugat-forbindelsen.Preparation of the Peptide-Hemocyanin Conjugate Compound.
100 mg hæmocyanin (dialyseret mod 5x31 vand 35 og herefter lyophiliseret) opløses i 3 ml vand, og hertil sættes 24 mg af peptidet I-C-E-S-C-P-T-G-G-Q-N-Y-S-P.Dissolve 100 mg of hemocyanin (dialyzed against 5x31 water 35 and then lyophilized) in 3 ml of water and to this add 24 mg of the peptide I-C-E-S-C-P-T-G-G-Q-N-Y-S-P.
5 DK 168872 B1 o5 DK 168872 B1 o
Under omrøring ved stuetemperatur sættes hertil i løbet af 2 timer portionsvis i alt 1 g N-cyclohexyl-N'-/£-(4-morpholinyl)ethyljcarbodiimid-methyl-p-toluensulfonat.With stirring at room temperature, a total of 1 g of N-cyclohexyl-N '- [beta - (4-morpholinyl) ethyl] carbodiimide methyl p-toluenesulfonate is added portionwise over 2 hours.
Efter omrøring natten over dialyseres reaktionsblandin-5 gen i 24 timer mod i alt 5 x 10 1 vand, og produktet isoleres derefter ved frysetørring. Der fås 137 mg af konju-gatforbindelsen.After stirring overnight, the reaction mixture is dialyzed for 24 hours against a total of 5 x 10 1 water and the product is then isolated by freeze-drying. 137 mg of the conjugate compound is obtained.
Eksempel 2 10Example 2 10
Radioimmunbindingsanalyse.Radio immune binding assay.
300 ^ul antiserum fra kaniner, der er immuniseret med det ifølge eksempel 1 fremstillede konjugat, i en egnet fortynding inkuberes natten over med 100 ^ul af 15 en prokollagen peptid (type III)-opløsning (1 ng prote-in/100 ^ul, fremstillet som det er beskrevet i europæisk patentskrift nr. 4940, eksempel 1), der er radioaktivt 125 mærket med I. De dannede antigen-antistof-komplekser udfældes ved tilsætning af et antiserum fra en anden dyre-20 art, hvilket er rettet mod immunoglobulin G fra den til immuniseringen anvendte dyreart. Efter centrifugering og dekantering af den overstående væske bestemmes mængden af udfældet radioaktivitet i et 7*- scintillationsspektro-meter.300 µl antiserum from rabbits immunized with the conjugate prepared according to Example 1 in a suitable dilution is incubated overnight with 100 µl of a procollagen peptide (type III) solution (1 ng protein / 100 µl , prepared as described in European Patent No. 4940, Example 1), which is radiolabeled with I. The antigen-antibody complexes formed are precipitated by the addition of an antiserum of another animal species, which is directed to immunoglobulin G from the animal species used for immunization. After centrifuging and decanting the supernatant, the amount of precipitated radioactivity is determined in a 7 * scintillation spectrometer.
2525
Eksempel 3 Radloimmunanalyse.Example 3 Radloimmune Assay.
0,2 ml af den prøve, der skal analyseres, eller 30 af prokollagen peptid (type III)-standarden inkuberes med en hvad angår mængden af mærket antigen begrænsende mængde af antiserum (i 0,1 ml puffer) natten over ved 4°C.0.2 ml of the sample to be analyzed or 30 of the procollagen peptide (type III) standard is incubated with an amount of labeled antigen limiting amount of antiserum (in 0.1 ml buffer) overnight at 4 ° C .
125125
Efter tilsætning af 0,1 ml I-mærket prokollagen peptid (type III) (indeholder 1 ng protein) inkuberes der i 35 6-8 timer ved 4°C, De dannede antigen-antistof-komplek ser udfældes ved hjælp af et mod IgG fra den til immuni- 6 DK 168872 B1After the addition of 0.1 ml of I-labeled procollagen peptide (type III) (containing 1 ng of protein), incubate for 6-8 hours at 4 ° C. The antigen-antibody complexes formed are precipitated by an anti-IgG from the to immune 6 DK 168872 B1
OISLAND
seringen anvendte dyreafct rettet antiserum. Efter fracen-trifugering og dekantering af den overstående væske bestemmes den udfældede radioaktivitet i et T-scinttila-tionsspektrometer.the animal used animal effect directed antiserum. After fractionation and decanting of the supernatant, the precipitated radioactivity is determined in a T-scintillation spectrometer.
5 Ved sammenligning med en standardkurve, til frem stillingen af hvilken standarder med forskellige koncentrationer af prokollagen peptid (type III) er anvendt, kan koncentrationen af prokollagen peptid (type III) derefter bestemmes i opløsningen med den ukendte koncentration.By comparison with a standard curve, for the preparation of which standards with different concentrations of procollagen peptide (type III) are used, the concentration of procollagen peptide (type III) can then be determined in the solution of the unknown concentration.
1010
Eksempel 4Example 4
Bestemmelse af molvægtfordelingen for med antistofferne 15 ifølge opfindelsen reagerende antigener i serum fra f.eks. okse og svin.Determination of the molecular weight distribution of serum antigens reacting with the antibodies of the invention from e.g. beef and pigs.
1 ml serum fraskilles ved gelfiltrationskromato-grafi via en "Sephacryl S 300"-søjle (1,6 x 130 cm), der er ækvilibreret i fosfatpuffret saltvand (PBS) 20 med 0,04 % Tween 20. Hver 0,2 ml af fraktionerne (hver 2,8 ml) anvendes til radioimmunanalysen ifølge eksempel 3.1 ml of serum is separated by gel filtration chromatography via a "Sephacryl S 300" column (1.6 x 130 cm) equilibrated in phosphate buffered saline (PBS) 20 with 0.04% Tween 20. Each 0.2 ml of the fractions (each 2.8 ml) are used for the radioimmunoassay of Example 3.
Fig. 1a viser elueringsprofilen af antigenet fra svineserum, fig. 1b viser den af antigenet fra okseserum i 25 sammenligning med profilen af det ifølge europæisk patentskrift nr. 4940 bestemte antigen. Toppene 1/1a svarer til intakt prokollagen type III eller pN-kollagen type III (prokollagen, der mangler den C-terminale ende), toppene 2/2a svarer til intakt aminoterminal prokollagen 30 peptid type III, toppene 3/3a svarer til Col 1 samt til nedbrydningsprodukter af det aminoterminale prokollagen peptid type III med samme molvægt som Col 1.FIG. Figure 1a shows the elution profile of the pig serum antigen; 1b shows the bovine serum antigen in comparison with the profile of the antigen determined by European Patent No. 4940. Peaks 1 / 1a correspond to intact procollagen type III or pN collagen type III (procollagen lacking the C-terminal end), peaks 2 / 2a correspond to intact amino-terminal procollagen 30 peptide type III, peaks 3 / 3a correspond to Col 1 as well as for degradation products of the amino terminal procollagen peptide type III of the same molecular weight as Col 1.
3535
Claims (11)
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| DE3714634 | 1987-05-02 | ||
| DE19873714634 DE3714634A1 (en) | 1987-05-02 | 1987-05-02 | METHOD FOR SELECTIVE IMMUNOLOGICAL DETERMINATION OF INTACT PROCOLLAGEN PEPTIDE (TYPE III) AND PROCOLLAGEN (TYPE III) IN BODY LIQUIDS AND MEANS TO IMPLEMENT IT |
Publications (3)
| Publication Number | Publication Date |
|---|---|
| DK232988D0 DK232988D0 (en) | 1988-04-28 |
| DK232988A DK232988A (en) | 1988-11-03 |
| DK168872B1 true DK168872B1 (en) | 1994-06-27 |
Family
ID=6326686
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| DK232988A DK168872B1 (en) | 1987-05-02 | 1988-04-28 | Method for producing antibodies for immunological determination of amino-terminal procollagen peptide (type III) and / or procollagen (type III), antibodies produced by the method, use of the antibodies for immunological determination of amino-terminal procollagen peptide (type III) and / or procollagen (type III) and starting product for use in the process |
Country Status (10)
| Country | Link |
|---|---|
| EP (1) | EP0298210B1 (en) |
| JP (1) | JPH0780913B2 (en) |
| AT (1) | ATE98377T1 (en) |
| DE (2) | DE3714634A1 (en) |
| DK (1) | DK168872B1 (en) |
| ES (1) | ES2061545T3 (en) |
| FI (1) | FI95579C (en) |
| IE (1) | IE63385B1 (en) |
| NO (1) | NO175639C (en) |
| PT (1) | PT87375B (en) |
Families Citing this family (12)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6010862A (en) * | 1987-11-06 | 2000-01-04 | Washington Research Foundation | Methods of detecting collagen type III degradation in vivo |
| DE4225038C2 (en) * | 1992-07-29 | 1995-11-30 | Boehringer Mannheim Gmbh | Production and use of antibodies against collagen |
| DK0711415T3 (en) * | 1993-07-28 | 1999-04-26 | Roche Diagnostics Gmbh | Immunoassay for detection of collagen or collagen fragments |
| GB9506050D0 (en) | 1995-03-24 | 1995-05-10 | Osteometer A S | Assaying collagen fragments in body fluids |
| ATE199185T1 (en) | 1994-10-17 | 2001-02-15 | Osteometer Biotech As | ASSESSMENT OF FRAGMENTATION PATTERNS OF COLLAGEN IN BODY FLUID AND DIAGNOSIS OF DISORDERS RELATED TO COLLAGEN METABOLISM |
| US6107047A (en) * | 1996-03-21 | 2000-08-22 | Osteometer Biotech A/S | Assaying protein fragments in body fluids |
| GB9617616D0 (en) | 1996-08-22 | 1996-10-02 | Osteometer Biotech As | Assaying protein fragments in body fluids |
| US6660481B2 (en) | 1996-12-09 | 2003-12-09 | Osteometer Biotech A/S | Sandwich assays for collagen type I fragments |
| US6117646A (en) * | 1997-09-22 | 2000-09-12 | Osteometer Biotech A/S | Assaying protein fragments in body fluids |
| US7541149B1 (en) | 1998-05-28 | 2009-06-02 | Siemens Healthcare Diagnostics, Inc. | Monoclonal antibody and assay for detecting PIIINP |
| US6602980B1 (en) | 1998-06-19 | 2003-08-05 | Washington Research Foundation | Collagen type III synthetic peptides for collagen resorption assays |
| US6916903B2 (en) | 1998-06-19 | 2005-07-12 | Washington Research Foundation | Collagen type III synthetic peptides for collagen resorption assays |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| DE2816841A1 (en) * | 1978-04-18 | 1979-10-31 | Max Planck Gesellschaft | RADIOIMMUNOLOGICAL DETERMINATION OF PROCOLLAGEN (TYPE III) AND PROCOLLAGEN PEPTIDE (TYPE III) |
| DE3209149A1 (en) * | 1982-03-13 | 1983-10-06 | Hoechst Ag | METHOD FOR THE COMMON IMMUNOLOGICAL DETERMINATION OF PROCOLLAGEN PEPTIDE (TYPE III) AND PROCOLLAGEN PEPTIDE COL 1 (TYPE III) AND METHOD FOR THE PRODUCTION OF ANTI-PROCOLLAGEN PEPTIDE COL 1 (TYPE III) SERUM |
-
1987
- 1987-05-02 DE DE19873714634 patent/DE3714634A1/en not_active Withdrawn
-
1988
- 1988-04-27 DE DE88106747T patent/DE3886109D1/en not_active Expired - Fee Related
- 1988-04-27 AT AT88106747T patent/ATE98377T1/en not_active IP Right Cessation
- 1988-04-27 ES ES88106747T patent/ES2061545T3/en not_active Expired - Lifetime
- 1988-04-27 EP EP88106747A patent/EP0298210B1/en not_active Expired - Lifetime
- 1988-04-28 JP JP63104474A patent/JPH0780913B2/en not_active Expired - Lifetime
- 1988-04-28 DK DK232988A patent/DK168872B1/en not_active IP Right Cessation
- 1988-04-29 IE IE128988A patent/IE63385B1/en not_active IP Right Cessation
- 1988-04-29 NO NO881904A patent/NO175639C/en not_active IP Right Cessation
- 1988-04-29 PT PT87375A patent/PT87375B/en active IP Right Grant
- 1988-04-29 FI FI882018A patent/FI95579C/en not_active IP Right Cessation
Also Published As
| Publication number | Publication date |
|---|---|
| IE63385B1 (en) | 1995-04-19 |
| NO881904L (en) | 1988-11-03 |
| NO881904D0 (en) | 1988-04-29 |
| FI882018A0 (en) | 1988-04-29 |
| EP0298210A2 (en) | 1989-01-11 |
| FI882018A (en) | 1988-11-03 |
| NO175639C (en) | 1994-11-09 |
| JPS63292061A (en) | 1988-11-29 |
| PT87375B (en) | 1992-08-31 |
| EP0298210B1 (en) | 1993-12-08 |
| EP0298210A3 (en) | 1991-05-15 |
| DK232988A (en) | 1988-11-03 |
| JPH0780913B2 (en) | 1995-08-30 |
| IE881289L (en) | 1988-11-02 |
| DE3886109D1 (en) | 1994-01-20 |
| DK232988D0 (en) | 1988-04-28 |
| NO175639B (en) | 1994-08-01 |
| FI95579C (en) | 1996-02-26 |
| DE3714634A1 (en) | 1988-11-17 |
| ATE98377T1 (en) | 1993-12-15 |
| ES2061545T3 (en) | 1994-12-16 |
| PT87375A (en) | 1989-05-31 |
| FI95579B (en) | 1995-11-15 |
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Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| B1 | Patent granted (law 1993) | ||
| PBP | Patent lapsed |