CA1338324C

CA1338324C - Monoclonal antibody for the selective immunological determination of intact procollagen peptide (type iii) and procollagen (type iii) in body fluids

Info

Publication number: CA1338324C
Application number: CA000597887A
Authority: CA
Inventors: Dietrich Brocks; Volkmar Gunzler-Pukall; Henning Hachmann; Jurgen Punter; Rupert Timpl
Original assignee: Hoechst AG
Current assignee: Sanofi Aventis Deutschland GmbH
Priority date: 1988-04-27
Filing date: 1989-04-26
Publication date: 1996-05-14
Anticipated expiration: 2013-05-14
Also published as: EP0339443A3; PT90369A; NO891732D0; JPH07110880B2; FI98705C; PT90369B; DE58908787D1; IE66320B1; NO179107B; NO179107C; FI891953L; EP0339443B1; IE891372L; FI98705B; GR3014957T3; DK202189D0; FI891953A0; JPH0213396A; EP0339443A2; ES2065350T3

Abstract

It is possible with the aid of a monoclonal antibody which is specifically directed against an epitope of amino-terminal procollagen peptide (type III) which is not present on the fragment col 1, and of a second monoclonal or polyclonal antibody against an epitope of amino-terminal procollagen peptide (type III), to deter-mine the said peptide with great accuracy.

Description

`-` I 338324 ~, /, _ I

Description A monoclonal antibody for the selective immunological determination of intact procollagen peptide (type III) and procollagen (type III) in body fluid~

Procollagen peptide (type III) (P III P) is the amino-terminal ~lG~pLide of collagen (type III) which under-goes extracellular elimination after secretion of the procollagen (type III) molecule. Procollagen peptide (type III) in turn can be further cleaved with collagen-a~e to fragments col 1, col 2 and col 3, which can be isolated using methods of protein chemistry known per se (Nowack, H. et al., Eur. J. Biochem. 70, 205 - 216 (1976), Bruckner, P. et al., Eur. J. Biochem. 90, 595 -603 (1978)).

A radioimmunological determination method as described inEuropean Patent No. 4,940 (published 31 October 1979) can be used to determine the concentration of this procollagen peptide in body fluids. Knowledge of the serum concentration of the peptide provides information on the activity of fibrotic disorders, such as, for example, of the liver [Rohde, H. et al. Eur. J. Clin. Invest. 9, 451-459 (1979)]-An accurate selective determination of procollagen peptide (type III) and procollagen (type III) in serum and otherbody fluids has been proposed in European Patent Application 289,930 (published 09 November 1988). However, this entails the necessity for a number of centrifugation steps which make routine laboratory use of this test difficult. More straightforward manipulation is permitted by the immunoradiometric assay (IRNA) method. This method makes use of two antibodies, one of which is coupled to a solid carrier, which makes one precipitation step and thus one centrifugation and some pipetting steps re~nn~t.

~,.' , _ . , ~ .

~ - 2 - 1 338324 - A monoclonal antibody which reacts with a fragment of procollagen (type III) which has not hitherto been evident as a characteristic peak in the gel filtration chromatography has now, surprisingly, been found. This antibody, in combination with other monoclonal or poly-clonal antibodies with ~pecificity for procollagen peptide (type III) and/or procollagen (type III), permits the determination of these antigens using the IRMA
technique.

Hence the invention relates to:

1. A monoclonal antibody having the reaction pattern depicted in Fig. 2 towards intact procollagen (type III) and pN-collagen (procollagen lacking the C-terminal ~lo~e~Lide) (peak la), intact amino-_ 15 terminal procollagen peptide (type III) (peak 2a), degradation products of amino-terminal procollagen peptide (type III) whose molecular weights are between that of amino-terminal procollagen peptide (type III) and that of col 1 (peak 3a), as well as col 1 and degradation products of amino-terminal procollagen peptide (type III) with the same molecu-lar weight as col 1 (peak 4a).

2. A hybridoma cell line which is obt~in~ by fusion of cells from a myeloma line and lymphocytes from an animal previously immunized with procollagen peptide (type III) and which produces the antibody defined under 1.

3. A process for the preparation of the antibody defined under 1.

4. A method for the quantitative immunological de-termination of procollagen peptide (type III) using the antibody defined under 1.

5. A diagnostic composition for establishing the amount ~ _ 3 _ 1 338 324 of procollagen peptide (type III) in body fluids, which is composed of an effective amount of the monoclonal antibody defined under 1., alone or in combination with other antibodies, in particular with an effective amount of the monoclonal antibody proposed in European Patent Application 289,930, mixed with a carrier acceptable in diagnosis.

The invention is explained in detail hereinafter, especially the preferred embodiments. The invention is also defined in the patent claims.

The embodiments of the invention are hereby described in the drawings as follows:

Figure 1 shows the calibration plot of the immunoradiometric assay (BIT denotes the ratio of bound to total radioactivity employed). (See example 6) Figure 2 shows the elution profile of the antigen determined r using P III P 226 by comparison with the profile of the antigen determined with the aid of polyclonal antibodies (see example 7).

20 Figure 3 shows the elution profile of the antigen determined using P III P 296 by comparison with the profile of the antigen determined with the aid of polyclonal antibodies.

To prepare the monoclonal antibodies, it is possible~to immunize animals, preferably rodents such as, for example, 25 mice, rats, rabbits or guinea-pigs, with procollagen peptide (type III~ which has been isolated by the process of European Patent 4,940, in the presence of adjuvant. Mice are particularly preferably used, especially those of the SJL strain. The immune response is enhanced with repeated 30 booster injections, for example at an interval of 4 to 8 weeks. The success of the immunization is checked by ~~ - 3A - 1 338324 determining the concentration of antibodies in a radiological binding assay [R. Timpl and L. Risteli, Immunochemistry of the extracellular matrix, H. Furthmayr Ed., Vol. 1, 199 (1982)]. Some days before the fusion of the lymphocytes with a myeloma cell line the animals are treated with procollagen peptide (type III) without adjuvant. Lymphocytes are obtained from the animals and fused with a myeloma cell line, which can likewise originate from one of the abovementioned species, but preferably from the mouse, especially with the cell line P3X63AG8.653. It is advantageous to fuse lymphocytes with myeloma cell lines of the same species. The fusion and the further cultivation of the cell clones are carried out in a manner known to those skilled in the art, with the concentration of specific antibodies being determined in the supernatant from the cell cultures using an immunological binding assay. One clone is c ~

~ - 4 - 1338324 der-iving from this process. It is particularly preferable to use a cell line which is prepared by fusion of the mouse myeloma cell line P3X63AG8.653 with lymphocytes from mice of the SJL strain which have been immunized with procollagen peptide (type III), and which produces a monoclonal antibody having the reaction pattern shown in Fig. 2. This cell line was deposited on March 2, 1988, under the conditions of the R~l~Apest Treaty at the European Collection of Animal Cell Cultures (ECACC), PHLS
Centre for Applied Microbiology and Research, Porton Down, Salisbury, SP4 OJG, U.R., with the number 88030202.

The monoclonal antibodies according to the invention belong to the IgG, IgM and IgA protein classes. Anti-bodies of the IgG class, especially of the IgG2b sub-class, can be employed with particular advantage. The - properties of the monoclonal antibodies are illustrated using the monoclonal antibody PIIIP 226 obtAin~ from the cell line ECACC 88030202, when the antigens present in the serum are fractionated according to their molecular weight by gel filtration chromatography, and the frac-tions from the chromatography are employed in the radio-imunoassay. Fig. 2 shows the gel filtration chromato-graphy elution profile as shown by the monoclonal anti-body PIIIP 226 compared with the elution profile ex-hibited by polyclonal antibodies. Peak l/la corresponds to intact procollagen (type III) and pN-collagen (type III). Peak 2/2a corresponds to intact amino-terminal procollagen peptide (type III) and degradation products thereof with a similar size. Peak 3a covers degradation products of procollagen peptide (III) which produce in the chromatogram a peak which is smaller than that of procollagen peptide (type III) but distinctly larger than that of col 1. Thus the molecular weight of each of the degradation products is between about 45,000 (procollagen peptide) and about 10,000 (col 1). The peak 4/4a cor-responds to col 1 plus degradation products of the amino-terminal procollagen peptide (type III) with the same molecular weight as col 1. It emerges that the antigen s 1 338324 fraction which is also detected on analysis with poly-clonal antiho~ies and which has the molecular weight of the degradation product col 1, or is col 1, is detected distinctly more weakly by the monoclonal antibody accord-ing to the invention. The monoclonal antibody accordingto the invention thus has a specific action against an epitope of the amino-terminal procollagen peptide (type III) which is not present in col 1. Furthermore, some of the antigens detected by polyclonal antibodies in peak 2 are not recognized by the antibody PIIIP 226, 80 that, with the antibody PIIIP 226, two peaks (2a and 3a) are evident at the position of peak 2. Peak 3a i8 likewise not found using the antibody PIIIP 296 proposed in the German Patent Application No. DE P37 14 633.5, filed May 2, 1987 (granted as EP O 289 930 on April 27, 1988).

It is important for the preparation of the antibodies - according to the invention that a suitable source for obtAining the antigen is available. As already mentioned, human or animal, highly purified procollagen peptide (type III) is advantageously isolated by the process of European Patent 4,940, entailing tissue or pathological body fluids being degraded with collagenase, and the procollagen peptide being removed from the reaction solution and purified by a combination of chromatographic methods and/or imm~noA~orption.

The monoclonal antibody according to the invention can be used in a variety of immunological methods, including all types of radioimmunoassay, for example seguential satura-tion analysis or equilibrium analysis, as well as in other competitive and non-competitive binAing assays, such as fluorescence, enzyme, chemiluminescence or other immunoassays. It is especially suitable for use in sandwich methods in immunosorbent assays. The monoclonal antibody can therefore be employed in immunological methods for the isolation and characterization, as well as for the quantitative determination, of procollagen peptide (type III) in tissues and body fluids. The methods used are those known per se to those skilled in ~ - 6 - 1 338324 the art, advantageously entailing reaction of a liquid sample which contain~ procollagen peptide (type III) with the monoclonal antibody according to the invention, which is coupled to a solid matrix which is preferably composed of plastic material, in particular plastic tubes, and determination of the amount of procollagen peptide (type III) by bin~i~g of a polyclonal or of a second monoclonal antibody, preferably of the monoclonal antibody proposed in European Patent Application 289,930, which is provided with a radioactive or other tracer. This method can also be carried out by binding polyclonal or monoclonal antibodies, especially the monoclonal antibody proposed in European Patent Application 289,930, to a solid matrix and bringing about reaction with the antigen, with this antigen then being determined quantitatively by bi n~ i ng of the labeled antibody according to the invention. In _ these methods it is immaterial whether the procollagen peptide (type III) is still linke~ to the amino terminus of procollagen (type III) or not. The degradation pro-ducts of procollagen peptide (type III), e6pecially col 1, which have hitherto interfered with the immunological determination using polyclonal antih~ie~ are not detected by the assay system described.
.
The monoclonal antibody proposed in European Patent Application 289,930 exhibits the reaction pattern depict-ed in Figure 3 towards intact procollagen (type III) and pN-collagen (procollagen lacking the C-terminal ~o~ep-tide, peak 4a), intact amino-terminal procollagen peptide (type III) (peak 5a) as well as col 1 and degradation products of amino-terminal procollagen peptide (type III) with the same molecular weight as col 1 (peak 6a).

The procedure for the preparation of this monoclonal antibody is essentially that described above. It is particularly preferred to use a cell line prepared by fusion of the mouse myeloma cell line P3X63AG8.653 with lymphocytes from mice of the SJL strain immunized with procollagen peptide (type III). This cell line is ~- _ 7 _ 1 338 324 deposited at the European Collection of Animal Cell Cultures (ECACC), PHLS Centre for Applied Nicrobiology and Research, Porton Down, Salisbury, SP4 OJG, U.R. under the number 87042308.

The invention is explained further in the examples which follow. Unless otherwise indicated, percentage data relate to weight.

Fxample 1 Preparation of procollagen peptide (type III) Procollagen peptide (type III) is prepared by the action of collagenase on procollagen (type III) at 37C. The peptide i8 exposed to no denaturing agents during this.
_ A modified process is used to prepare larger amounts of the peptide. All process steps up to the action of collagenase are carried out in a cold room. The various NaCl solutions employed for solubilization contain 0.05 M Tris-HCl, pH 7.4, 0.01 N EDTA, sodium azide (200 mg/ml) and the protease inhibitors phenylmethylsulfonyl fluoride (3~g/ml) and p-chloromercuribenzoate (3~g/ml).

Fetal calf skin (3 kg) is homogenized and extracted for two days in 10 1 of 1 M NaCl. Dissolved collagen is precipitated from the extraction solution by addition of solid NaCl to a final concentration of 2.5 M. After the precipitate has been stirred overnight it is collected by centrifugation (1800 x g, 20 minutes), washed twice with 2.5 M NaCl and redissolved by stirring it overnight in 10 1 of 0.5 N NaCl. Small amounts of insoluble material are removed by centrifugation. The mixture of collagen (type III) and procollagen (type III) obtained in this way is precipitated with 1.6 M NaCl. The precipitate is then suspen~^~ in 2 1 of O.05 N Tris-HCl (pH 8.0) and, after addition of 0.02 M CaCl2, heated at 50C for 20 minutes and subsequently incubated with 1500 U of bac-terial collagenase (CLSPA, Worthington, USA) per gram of ~ 8 - 1 338324 moist precipitate at 37C for 3 hour3. After the col-lagenase has acted, the precipitate which has formed is removed by centrifugation, and the solution is dialyzed against 0.005 M Tris-HCl, pH 8.0, 6.8 M urea and passed through a DEAE-cellulose column (5.0 x 30 cm) which has been equilibrated with the same buffer.

The proteins bound to the column are washed out with an NaCl solution who~e concentration rises from 0 to 0.3 M.
The total amount eluted is 2 1. The solution flowing out of the column is examined for the absorption at 236 nm and for its antigenic activity by use of antihoA ies specific for the amino-terminal segment of procollagen (type III). Normally it is the last peak eluted from the column which contains the procollagen peptide ~type III).
Dialysis Ag~ in~t distilled water removes salts from the _ peptide, which is lyophilized. Subsequent purification is carried out on a column cont~i n i ng agarose A 1.5 ~ (2 x 120 cm) (from Biorad) which is equilibrated with 1 M
CaCl2, 0.05 M Tris-HCl, pH 7.5.

~z~ ple 2 ~ Hybridoma preparation Mice of the SJL strain are immunized intramuscularly with 5 ~g of procollagen peptide (type III), obt~in^~ as in Example 1, in the ~ n~ of complete Freund's ad~uvant.
The immune ~onse is qnh~nce~ after four weeks and after three months by another intramuscular in~ection of 5 ~g of procollagen peptide (type III) in the presence of incomplete F~eund's ad~uvant. Three days before the fusion the immune e_~onse is enh nced by in~ection of a further 50 ~g of procollagen peptide (type III).

For the fusion, the animals are sacrificed, and the spleen cells are isolated. The spleen cells are fused with the yeloma cell line P3X63AG8.653 in the presence of polyethylene glycol. (The resulting cell line is _ deposited at the ECACC under the number 88030202.) Spleen cell x P3X63AG8.653 hybrid~ are selected by cultivation of the fusion mixture in hypoxanthine/aminopterin/thymid-ine medium for a period of two weeks. The resulting cell clones are subcloned several times to obtain a monoclonal cell line. The ~upernatants of the resulting cell colon-ies are assayed for antibody production in a radio-immunological b1n~1n~ assay. The bimonoclonal antibody PIIIP 226 is obtAine~ in this way.

~ample 3 Radioimmunological bi n~i n~ assay 300 ~1 of cell culture supernatant, or another sample such a8, for example, ascites after cultivation of _ hybridoma cells in the abdominal cavity of mice, are incubated with 100 ~1 of a 125I-procollagen peptide (type III) ~olution (1 ng of protein/100 ~ 1, prepared as described in EP 4,940, Example 1) overnight. The antigen-antibody complexes which are formed are precipitated by addition of anti-mouse IgG serum from sheep or another species. Centrifugation and removal of the supernatant by ~ ~ec~ntation are followed by determination of the amount of precipitated radioactivity in a gamma scintillation ~pectrometer.

~ample 4 Radioactive labeling of the antibodie~

0.2 ml of a solution contA; n; ng 0.2 mg of the monoclonal antibody PIIIP 296 obtained as described in German Patent Application P 3,714,633.5 (priority date 10 July 1987), Example 2, or of another antibody, in 0.05 N phosphate buffer, pH 7.4, are placed in a polystyrene assay tube (12 x 55 mm), and 100 Nsq of Na12sI solution buffered with 10 ~1 of 0.5 M phosphate buffer, pH 7.4, are added. Addition of 50 ~1 of an aqueous solution of 20 ~g of chloramine T is followed by lo - 1 338324 mixing for 1 minute. The iodination reaction is subse-quently stopped by addition of 50 ~1 of an aqueou~
solution of 20 ~g of sodium disulfite. The unreacted Na125I is subsequently removed from the 125I-labeled anti-body by chromatography on an anion exchanger. The chro-matographic fractions which contain the purified 125I-labeled antibody are diluted with a ~olution of 20 g of Tween 20 and 14.6 g of Na2EDTA in one liter of 0.05 M
Tris-HCl, pH 8.0, 80 that the concentration of the labeled antibody is 200 ~g/l.

Exa-ple S

Coating of assay tubes with the antihoAies To immobilize antihoA ies on poly~ylel~e assay tubes (12 _ x 75 mm) 300 ~1 of a solution of 4 mg/l antibody, for example PIIIP 226, in 0.01 N sodium phosphate buffer, pH

6.4, are placed in each tube and incubated at room temperature overnight. The antibody solution is then removed by aspiration, and 500 ~1 of a 1 % strength solution of bovine serum albumin in 0.05 M Tris-citrate, pH 7.5, are placed in each tube. After incubation at room temperature overnight the solution is removed by aspir-ation. The antibody-coated tubes are dried over silica gel.

~a-ple 6 I_ unoradiometric assay (radioimmunometric assay, IRMA) 0.1 ml of the sample for analysis, or of procollagen peptide (type III) stAn~rd, is incubsted in poly_~y ene assay tubes which have been coated with the monoclonal antibody PIIIP 226 as in Example 5, with the addition of 0.1 ml of phosphste-buffered saline (PBS) at room temper-ature for 2 hours. The assay tubes are subsequently washed twice with 1 ml of PBS each time. Then 200~ 1 of 125I-labeled antibody PIIIP 296 (= 40 ng of antibody), or of another antibody, are placed in the tubes and incu-bated at room temperature for 2 hours. The radioactivity of the antibody-antigen-l25I-antibody complexes bound to the tube wall is determined, after two washings with 1 ml of PBS each time and subsequent decantation, in a gamma scintillation spectrometer.

It is then possible to calculate the concentration of procollagen peptide (type III) in the unknown sample solution by comparison with a calibration plot which has been constructed by employing st~n~Ards contA;ning different amounts of procollagen peptide (type III). Fig.
1 shows the cAl~hration plot of the immunoradiometric assay (B/T denotes the ratio of bound to total radio-activity employed).

- 15 E~ample 7 Determination of the molecular weight distribution of the antigens in human serum which react with PIIIP 226 1 ml of serum is fractionated by gel filtration chroma-tography on an allyldextran crosslinked with N,N'-methyl-enebisacrylamide t-Sephacryl S 300 column (1.6 x 130 cm)], equilibrated in PBS contA~n1~g 0.04 % of a nonionic detergent, for example a polyethoxylated sorbitan mono-laurate (Tween 20). 0.2 ml of each individual fraction is incubated with the antibody to be investigated, in an amount which is limiting with regard to the amount of labeled antigen (in 0.1 ml of buffer), and with 0.1 ml of 125 I-labeled procollagen peptide (type III) (contains 1 of protein) at 4C overnight. It is 81~hA ~uently incubated with a previously assayed amount of anti-mouse IgG serum from sheep in the presence of 10 % polyethylene glycol (PEG 6000) for 1 hour. The precipitated antigen-antibody complexes are spun down (1500 x g) and, after decanta-tion, the rA~oActivity is determined in a gamma scintil-lation spectrometer. It is then possible to determine, by 35 ~ comparison with a calibration plot which has been .

. .

constructed by employing st~n~rds cont~i~ing different amounts of procollagen peptide (type III), the concentra-tion in the chromatography fractions of antigens which react with the antibody employed. Fig. 2 shows the elution profile of the antigen determined using PIIIP 226 by comparison with the profile of the antigen determined with the aid of polyclonal antibodies.

Claims

1. A monoclonal antibody having the reaction pattern depicted in Fig. 2 and characterized as having (a) affinity to intact procollagen (type III), pN-collagen (peak 1a), and intact amino-terminal procollagen peptide (type III) (peak 2a); (b) low affinity to degradation products of amino-terminal procollagen peptide (type III) whose molecular weights are between that of amino-terminal procollagen peptide (type III) and that of col 1 (peak 3a); and (c) no affinity to col 1 and to degradation products of amino-terminal procollagen peptide (type III) with the same molecular weight as col 1 (peak 4a).

2. A monoclonal antibody as claimed in claim 1, having a specific action against an epitope of amino-terminal procollagen peptide (type III) which is not present in col 1.

3. A monoclonal antibody as claimed in claim 1, which belongs to the IgG class.

4. A monoclonal antibody as claimed in claim 2, which belongs to the IgG class.

5. A monoclonal antibody as claimed in claim 1, which is formed by a hybridoma which is produced by fusion of cells of a myeloma line with lymphocytes from an animal which has previously been immunized with amino-terminal procollagen peptide (type III).

6. The monoclonal antibody PIIIP 226 of the hybridoma cell line ECACC 88030202.

7. A hybridoma cell line which produces an antibody as claimed in any one of claims 1 to 6, formed by fusion of cells from a myeloma cell line and of lymphocytes from an animal which has previously been immunized with procollagen peptide (type III).

8. The hybridoma cell line ECACC 88030202.

9. A process for the preparation of a monoclonal antibody as claimed in claim 1, which comprises:
a. animals being immunized with amino-terminal procollagen peptide (type III);
b. lymphocytes being obtained and fused with myeloma cells;
c. the hybrids being selected for the presence of an antibody having the propertieæ indicated in claim 1, and being cloned; and d. the antibody being obtained from the said clones.

10. The process as claimed in claim 9, wherein the hybridoma cell line ECACC 88030202 is employed to carry out step d).

11. A method for the quantitative immunological determination of procollagen peptide (type III) using antibodies, which comprises:
a. a liquid sample which contains amino-terminal procollagen peptide (type III) or procollagen (type III) being reacted with the monoclonal antibody as claimed in any one of claims 1 to 6, and b. the amount of amino-terminal procollagen peptide (type III) or of procollagen (type III) being determined via the antigen-antibody complex formed.

12. A method for the quantitative immunological determination of procollagen peptide (type III) using antibodies, which comprises:

a. the monoclonal antibody as claimed in claim 1 being coupled to a solid matrix;

b. a liquid sample which contains amino-terminal procollagen peptide (type III) or procollagen (type III) being reacted with the said antibody; and c. the bound antigen being detected by labeled monoclonal or polyclonal antibodies with specificity for procollagen peptide (type III) or procollagen (type III).

13. The method as claimed in claim 12, wherein monoclonal antibodies as claimed in claim 1 are employed in step a), and the labeled monoclonal antibody having the reaction pattern depicted in Fig. 2 towards intact procollagen (type III) and pN-collagen (peak 1a), intact amino-terminal procollagen peptide (type III) (peak 2a), degradation products of amino-terminal procollagen peptide (type III) whose molecular weights are between that of amino-terminal procollagen peptide (type III) and that of col 1 (peak 3a), as well as col 1 and degradation products of amino-terminal procollagen peptide (type III) with the same molecular weight as col 1 (peak 4a) is employed in step c).

14. The method as claimed in claim 12, wherein the monoclonal antibody employed in step a) is the monoclonal antibody as claimed in claim 1 and wherein the monoclonal antibody employed in step c) is an antibody which has the reaction pattern depicted in Figure 3 towards intact procollagen (type III) and pN-collagen (peak 4a), intact amino-terminal procollagen peptide (type III) (peak 5a), as well as col 1 and degradation products of amino-terminal procollagen peptide (type III) with the same molecular weight as col 1 (peak 6a).

15. The method as claimed in claim 13, wherein the monoclonal antibody employed in step a) is the antibody which has the reaction pattern depicted in Figure 3 towards intact procollagen (type III) and pN-collagen (peak 4a), intact amino-terminal procollagen peptide (type III) (peak 5a) as well as col 1 and degradation products of amino-terminal procollagen peptide (type III) with the same molecular weight as col 1 (peak 6a).

16. The method as claimed in any one of claims 14 or 15, wherein the monoclonal antibody employed is PIIIP 296 of the hybridoma cell line ECACC 87042308.

17. A diagnostic composition for establishing the amount of procollagen peptide (type III) in body fluids, which contains an effective amount of the monoclonal antibody as claimed in any one of claims 1 to 6, alone or in combination with other antibodies, mixed with a carrier acceptable in diagnosis.