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IE881289L - A method for the selective immunological determination of¹intact procollagen peptide (Type III) and procollagen (Type¹III) in body fluids, and means for carrying it out - Google Patents

A method for the selective immunological determination of¹intact procollagen peptide (Type III) and procollagen (Type¹III) in body fluids, and means for carrying it out

Info

Publication number
IE881289L
IE881289L IE881289A IE128988A IE881289L IE 881289 L IE881289 L IE 881289L IE 881289 A IE881289 A IE 881289A IE 128988 A IE128988 A IE 128988A IE 881289 L IE881289 L IE 881289L
Authority
IE
Ireland
Prior art keywords
peptide
type iii
procollagen
antibodies
bound
Prior art date
Application number
IE881289A
Other versions
IE63385B1 (en
Inventor
Dietrich Brocks
Rupert Timpl
Original Assignee
Max Planck Gesellschaft
Hoechst Ag
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Max Planck Gesellschaft, Hoechst Ag filed Critical Max Planck Gesellschaft
Publication of IE881289L publication Critical patent/IE881289L/en
Publication of IE63385B1 publication Critical patent/IE63385B1/en

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    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K14/00Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/78Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin or cold insoluble globulin [CIG]
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K16/00Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00Medicinal 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)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Toxicology (AREA)
  • Zoology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Immunology (AREA)
  • Peptides Or Proteins (AREA)
  • Investigating Or Analysing Biological Materials (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)
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Abstract

It is possible by immunising animals with a peptide of the sequence I-C-E-S-C-P-T-G-G-Q-N-Y-S-P bound to an immunogenic protein to raise antibodies which can be used to determine procollagen peptide (type III) and procollagen type III selectively in body fluids.

Description

63385 —!*-" HOECHST AKTIENGESELLSCHAFT HOE 87/F 130 Dr. KH/rh Specification A aethod for the selective immunological determination of intact procollagen peptide (type III) and procollagen (type III) in body fluids, and means for carrying it out 5 Procollagen peptide (type III) is the amino-terminal propeptide of collagen (type III), which is cleaved off outside the cell after secretion of the procollagen (type III) molecule. The concentration of this procollagen peptide in body fluids can be determined using a radioimmunoIogica I 10 determination method as described in European Patent No. 4940. Knowledge of the serum concentration of the peptide allows conclusions to be drawn about the activity of fibro-tic disorders, such as; for example, of the liver CRohde, H. et al. Eur. J. Clin. Invest. 9_, 451 - 459 ( 1979)]. 15 However, accurate selective determination of procollagen peptide (type III) in serum and other body fluids is not possible using the methods hitherto described, because the polyclonal antibodies which are used in these methods react, with different, lower affinity, with various antigens which 20 occur in serum and some of which are breakdown products of procollagen peptide (type III) [Niemela, 0. et al. Clin.
Chim. Acta 124, 39 - 44 (1982)]. The result of this is that the serum dilution plots and the dilution plots of other body fluids are not parallel to the calibration plot 25 constructed using pure procollagen peptide (type III), and hence it is necessary to determine the antigen content in several dilutions of each unknown sample in order to establish the antigen concentration via the 50 % intercept on the dilut ion plot. 30 Another disadvantage of these methods is that, because the cross-reactivity of the antibodies with rat or mouse antigen is too low, no determination of the antigen concentration - 2 - in body fluids from these species has been possible.
It is possible to determine the concentrations of the antigens in rat serum using the method described by Schuppan 5 et al. [J. Hepatol. 3, 27-37 (1986)]. However, this method has the same disadvantage as that described for the method for human sera [Niemeta et al. Clin. Chim. Acta 12 4, 39-44 (1982)] that the inhibition plots are not parallel for different body fluids. 10 This technical problem can be solved using the method of European Patent Application 0,089,008, in which the antibodies used have comparable affinities for intact procollagen peptide (type III) and its breakdown product Col 1. This method determines intact and degraded procollagen 15 peptide (type III) together, but this results in imprecision in the diagnostic conclusions because the normal popu-ulation and the patient population may overlap greatly.
It has now been found, surprisingly, that when a peptide with a defined amino acid sequence is used for immunization 20 it is possible to obtain antibodies with whose aid specific determination of intact procollagen peptide (type III) is possible. Moreover, surprisingly, using these antibodies it is possible to determine the procollagen peptide (type III) content in body fluids of rats or mice, which 25 is of use for examining fibrosuppressive substances in animal experiments.
Thus the invention relates to a method for the immunological determination of amino-terminal procollagen peptide (type III) using antibodies, which comprises 30 a) immunization of animals with a peptide of the sequence I-C-E-S-C-P-T-G-G-Q-N-Y-S-P, bound to an immunogenic protein, b) obtaining from the serum the antibodies which react with intact amino-terminal procollagen peptide (type III) and c) determination of the amount of amino-terminaI procollagen peptide (type III) and of procollagen (type III) via the antigen-antibody complex which is formed.
The invention also relates to antibodies which are raised by immunization of animals with the abovementioned peptide which is bound to an immunogenic protein.
The invention is described in in its preferred embodiments, ined in the claims. detail hereinafter, especially The invention is also def- For the preparation of the antibodies, animals, preferably rodents such as, for example, rabbits or guinea pigs, or goats and sheep are immunized with the peptide of the sequence I-C-E-S-C-P-T-G-G-Q-N-Y-S-P coupled to a suitable immunogenic protein in the presence of complete adjuvant. Rabbits are particularly preferably used. The immune response is enhanced by repeated booster injections, for example at intervals of 4 to 8 weeks. The success of immunization is checked by determining the concentration of antibodies in a radioimmunoLogical binding assay [R. Timpl and L. Risteli, Immunochemistry of the extracellular matrix, H. Furthmayr Ed., Vol. 1, 199 (1982)3.
All immunogenic proteins are suitable as proteins to which the said peptide can be bound. Hemocyanin, albumin or polylysine are preferably used. The peptide can be prepared by methods known to the expert, as described by, - 4 - for example, G. Barany and A.B. Merrifield in The Peptides Vol. 2 , pp 3-254 (1980), Academic Press, or E. Brown, R.C. Sheppard and B.J. Williams, J. Chem. Soc. Perkin Transact. _1, 1 161 ( 1983). 5 The antibodies according to the invention can be used as serum or purified in various immunological methods, including all types of radioimmunoassay, for example sequential saturation analysis or equilibrium analysis, labeling with chloramine T or Bo I ton-Hunter reagents [Felber, Meth. 10 Biochem. Anal. 22_, 1 ( 1974); Shelley et al., Clin. Chem. 19, 146 (1975)] as well as in other competitive binding assays such as fluorescence or enzyme immunoassays, chemi-luminescence or other immunoassays. The antibodies can thus be used in immunological methods for the isolation 15 and characterization and for the quantitative determination of procollagen peptide (type III) in tissues and body fluids. The procedure for quantitative determination makes use of methods known to the expert, by reacting a liquid sample which contains procollagen peptide (type III) with 20 the antibodies according to the invention, and determining the amount of procollagen peptide (type III) via the anti-gen-antibody complex which is formed. It is of no consequence for this whether the procollagen peptide (type III) is still linked to the amino terminus of procollagen (type 25 III) or not. The degradation products of procollagen peptide (type III), especially Col 1, which have hitherto interfered in the immunological determination, are not among the species detected by the antibodies according to the invention. 30 The invention is explained further in the examples which follow. Unless otherwise indicated, percentage data relate to weight.
Example 1 - 5 - Preparation of the peptide/hemocyanin conjugate compound 100 mg of hemocyanin (dialyzed against 5 x 3 I of water and then freeze-dried) are dissolved in 3 ml of water, and 5 24 mg of the peptide I-C-E-S-C-P-T-G-G-Q-N-Y-S-P are added-While stirring at room temperature, a total of 1 g of N-cyclohexyl-N'-C2-(4-morpholinyl)ethyl]carbodi imide-methyl— p-toIuenesuIfonate is added in portions over the course of 2 hours- The reaction mixture is stirred overnight and 10 then dialyzed against a total of 5 x 10 I of water for 24 hours, and the product is subsequently isolated by freeze-drying. 137 mg of the conjugate compound are obtained.
Example 2 15 RadioimmunoLogical binding assay 300 ul of antiserum from rabbits immunized with the conjugate prepared as in Example 1 are, in a suitable dilution, incubated overnight with 100 ul of a procollagen peptide type III solution (1 ng of protein/100 ul, prepared as de- 20 scribed in European Patent 4940, Example 1) which is radio- 12 5 actively labeled with I. The antigen-antibody complexes which are formed are precipitated by addition of an antiserum which is directed against immunoglobulin G of the species used for the immunization and is from a different 25 species. Following centrifugation and decantation of the supernatant, the amount of precipitated radioactivity is determined in a y~scinti 11 ation spectrometer.
Example 3 Rad io immunoassay 30 0.2 ml of the sample which is to be analyzed or of pro- - 6 - collagen peptide (type III) standard is incubated at 4°C overnight with an amount of antiserum (in 0.1 ml of buffer) which is limiting with respect to the amount of labeled 12 5 antigen. Addition of 0.1 ml of I-labeled procollagen 5 peptide (type III) (contains 1 ng of protein) is followed by incubation at 4°C for 6-8 hours. The antigen-anti-body complexes which are formed are precipitated by use of an antiserum directed against IgG of the species used for the immunization. Centrifugation and decantation of the 10 supernatant are followed by determination of the precipitated radioactivity in a y-scinti11 ation spectrometer.
It is then possible, by comparison with a calibration plot which has been constructed by using standards with different concentrations of procollagen peptide (type III), to 15 determine the concentration of procollagen peptide (type III) in the unknown solution.
Example 4 Determination of the molecular weight distribution of the antigens which react with the antibodies according to 20 the invention and are present in the serum of, for example, cattle and hogs 1 ml of serum is fractionated by gel filtration chromato- (R) graphy on a Sephacryl S 300 column (1.6 x 130 cm) equilibrated in phosphate-buffered saline, PBS, containing 25 0.04 % Tween 20. 0.2 ml of each of the fractions (2.8 ml each) is used in the radioimmunoassay of Example 3.
Fig. 1a shows the elution profile of the antigen from porcine serum, and Fig. 1b shows that of the antigen from bovine serum, comparing with the profile of the antigen 30 determined as in European Patent 4940. Peaks 1/1 a correspond to intact procollagen type III and pN collagen type III (procollagen lacking the C-terminal end); peaks 2/2a correspond to intact amino-terminal procollagen peptide type III; peaks 3/3a correspond to CoL 1 and breakdown products of amino-terminal procollagen peptide type III with the same molecular weight as Col 1.

Claims (4)

1. 1. Patent Claims: HOE 8 /F 130 1. A method for the immunological determination of amino-terminal procollagen peptide (type III) using antibodies, which comprises a) immunization of animals with a peptide of the sequence I-C-E-S-C-P-T-G-G-Q-N-Y-S-P bound to an immunogenic protein, b) obtaining from the serum the antibodies which react with intact amino-terminal procollagen peptide (type III) and c) determination of the amount of amino-terminal procollagen peptide (type III) and of procollagen (type III) via the antigen-antibody complex which is formed. I. The method as claimed in claim 1, wherein the peptide is bound to hemocyanin, albumin or polylysine. 5. The method as claimed in claim 1 or 2, wherein rodents or goats or sheep are immunized. ». The method as claimed in claim 3, wherein rabbits are immunized. i. Antibodies obtainable by immunization of animals with the peptide of the sequence I-C-E-S-C-P-T-G-G-Q-N-Y-S-P bound to an immunogenic protein. >. Antibodies as claimed in claim 5, wherein the peptide is bound to hemocyanin, albumin or polylysine. Antibodies as claimed in claim 5 or 6, wherein rodents or goats or sheep are immunized. Antibodies as claimed in claim 7, wherein rabbits are i mmun i z ed. A compound composed of the peptide of the sequence I-C-E-S-C-P-T-G-G-Q-N-Y-S-P bound to an immunogenic protein. The use of the compound claimed in claim 9 as an antigen. -lo11. A method according to Claim 1 for the immunological determination of amino-terminal procollagen peptide (type III) using antibodies, substantially as hereinbefore described and exemplified. j 1
2. An antibody according to Claim 5, substantially as * hereinbefore described and exemplified. 1
3. A compound according to Claim 9, substantially as hereinbefore described and exemplified. 1
4. Use according to Claim 10, substantially as hereinbefore described and exemplified. Dated this the 29th day of April, 1988 F. R. KELLY & CO. BY: Q lA. . , EXECUTIVE 27 Clyde Road, Ba^lsbridge, Dublin 4 AGENTS FOR THE APPLICANTS. J
IE128988A 1987-05-02 1988-04-29 A method for the selective immunological determination of intact procollagen peptide (Type III) and procollagen (Type III) in body fluids and means for carrying it out IE63385B1 (en)

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
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 (2)

Publication Number Publication Date
IE881289L true IE881289L (en) 1988-11-02
IE63385B1 IE63385B1 (en) 1995-04-19

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IE128988A IE63385B1 (en) 1987-05-02 1988-04-29 A method for the selective immunological determination of intact procollagen peptide (Type III) and procollagen (Type III) in body fluids and means for carrying it out

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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)

* Cited by examiner, † Cited by third party
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
FI960392L (en) * 1993-07-28 1996-01-29 Boehringer Mannheim Gmbh Immunoassay for the detection of collagen or collagen fragments
GB9506050D0 (en) 1995-03-24 1995-05-10 Osteometer A S Assaying collagen fragments in body fluids
ES2154739T3 (en) 1994-10-17 2001-04-16 Osteometer Biotech As ESTIMATION OF THE COLLAGEN FRAGMENTATION MODEL IN BODY FLUIDS AND THE DIAGNOSIS OF DISORDERS ASSOCIATED WITH THE 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
DE69705423T2 (en) 1996-12-09 2002-05-16 Osteometer Biotech As Herlev SANDWEIGHT TEST FOR DETECTING COLLAGEN FRAGMENTS
US6117646A (en) * 1997-09-22 2000-09-12 Osteometer Biotech A/S Assaying protein fragments in body fluids
DE69915059T2 (en) 1998-05-28 2004-08-26 Bayer Healthcare Ag MONOCLONAL ANTIBODY AND ASSAY FOR DETERMINING N-TERMINAL PROCOLLAGEN PROPEPTIDE TYPE III (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)

* Cited by examiner, † Cited by third party
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

Also Published As

Publication number Publication date
FI95579B (en) 1995-11-15
DE3886109D1 (en) 1994-01-20
FI882018A0 (en) 1988-04-29
NO881904L (en) 1988-11-03
EP0298210B1 (en) 1993-12-08
ATE98377T1 (en) 1993-12-15
DE3714634A1 (en) 1988-11-17
EP0298210A2 (en) 1989-01-11
PT87375A (en) 1989-05-31
JPS63292061A (en) 1988-11-29
DK232988A (en) 1988-11-03
DK168872B1 (en) 1994-06-27
NO881904D0 (en) 1988-04-29
FI95579C (en) 1996-02-26
PT87375B (en) 1992-08-31
FI882018L (en) 1988-11-03
NO175639B (en) 1994-08-01
ES2061545T3 (en) 1994-12-16
IE63385B1 (en) 1995-04-19
EP0298210A3 (en) 1991-05-15
NO175639C (en) 1994-11-09
JPH0780913B2 (en) 1995-08-30
DK232988D0 (en) 1988-04-28

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