IE881290L - Monoclonal antibodies for the selective immunological¹determination of intact procollagen peptide (Type III) and¹procollagen (Type III) in body fluids - Google Patents

Abstract

Procollagen peptide can be determined with great accuracy in body fluids with the aid of a monoclonal antibody which is specifically directed against an epitope of the amino-terminal procollagen peptide (type III) which is not present in Col 1.

Description

rv-d'1 HOECHST AKTIENGESELLSCHAFT Dr.KH/gm 87/F 131 Spec i f i c at i on Monoclonal antibodies for the selective immunological determination of intact procollagen peptide (type III) 5 and procollagen (type III) in body fluids ¥ Procollagen peptide (type III) is the amino-termina I propeptide of collagen (type III), which is cleaved off outside the cell after secretion of the procollagen (type 10 III) molecule. The concentration of this procollagen peptide in body fluids can be determined using a radio-immunological determination method as described in European Patent No. 4940. Knowledge of the serum concentration of the peptide allows conclusions to be drawn about 15 the activity of fibrotic disorders, such as, for example, of the liver CRohde, H. et al. Eur. J. Clin. Invest. 9_, 451 - 459 (1979)].

However, accurate selective determination of procollagen 20 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 occur in serum and some of which are break-25 down products of procollagen peptide (type III) CNiemela, 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 constructed using pure procollagen 30 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 dilution plot. ~ 35 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 - 2 - Col 1. This method determines intact and degraded procollagen peptide (type III) together, but this results in imprecision in the diagnostic conclusions because the normal population and the patient population may overlap 5 greatly.

Surprisingly, a monoclonal antibody which does not react with the breakdown products of procollagen peptide (type III) which occur in body fluids has now been found. Use 10 of this monoclonal antibody makes possible an immunological determination of the amino-1ermina I procollagen peptide (type III), which does not also determine its breakdown products and which is distinguished by serum dilution curves, the dilution curves of other body fluids 15 and the calibration curve showing complete parallelism.

Hence the invention relates to: 1. A monoclonal antibody having the reaction pattern 20 which is depicted in Figure 3 towards intact pro collagen (type III) and pN collagen (procollagen which is lacking the C-terminal propeptide, peak 4a), intact amino-terminal procollagen peptide (type III) (peak 5a), and Col 1 and breakdown products of the 25 amino-terminal procollagen peptide (type III) having the same molecular weight as Col 1 (peak 6a). 2. A hybridoma cell line which is formed by fusion of cells from a myeloma line and lymphocytes from an 30 animal which has previously been immunized with pro collagen peptide (type III), and which produces the antibodies described under 1. 3. A process for the preparation of the antibody des-35 c r ibed under 1. 4.

A method for the quantitative immunological determination of procollagen peptide (type III) using the antibody described under 1. - 3 - 5. A diagnostic composition for establishing the amount of procollagen peptide (type III) in body fluids, which is composed of an effective amount of the monoclonal antibody described in 1. , mixed with a dia- * 5 gnostically acceptable vehicle.

The invention is explained in detail hereinafter, especially the preferred embodiments. The invention is also defined in the patent claims. 10 For the preparation of the monoclonal antibodies, it is possible for animals, preferably rodents such as, for example, mice, rats, rabbits or guineapigs, to be immunized, in the presence of adjuvant, with procollagen pep-15 tide (type III) which has been isolated by the process of European Patent 4940. Mice are particularly preferably used, especially those of the SJL strain. The immune response is enhanced with repeated booster injections, for example at intervals of 4 to 8 weeks. The 20 success of immunization is checked by determination of the concentration of antibodies in a radioimmunoIogica I binding assay [R. Timpl and L. Risteli, Immunochemistry of the extracellular matrix, H. Furthmayr Ed., Vol. 1, 199 (1982)3. Some days before fusion of the lymphocytes 25 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 animal species, but is preferably 30 from the mouse, in particular with the cell line P3X63AG8. 653. It is advantageous to fuse lymphocytes with myeloma cell lines of the same species. The fusion and further cultivation of the cell clones are carried out in a manner known to the expert, with the concentration of spe-35 cific antibodies being determined in the supernatant from the cell culture using an immunological binding assay. Clones suitable for use in immunological methods are selected from the cell clones derived from the fusion.

It is particularly preferable to use a cell line which - 4 - is prepared by fusion of Lymphocytes from mice of the SJL strain against procoLLagen peptide type III with the mouse myeLoma ceLL line P3X63AG8.653. The Latter cell line is deposited at the European Collection of Animal • 5 Cell Cultures (ECACC), PHLS Centre for Applied Micro biology and Research, Porton Down, Salisbury, SP4 OJG, * U.K., under the number 87042308.

The monoclonal antibodies according to the invention be-10 long in the group of immunoglobulins, preferably in the class of IgG, IgA and IgM proteins. Antibodies of the IgG class, especially of the subclass IgG2b, can be used with particular advantage. The antibody according to the invention is particularly surprising because its 15 affinity to the antigen is higher than the affinity of polyclonal antibodies. The opposite is normally found. The properties of the monoclonal antibodies are illustrated by the monoclonal antibody PIIIP 296, which is obtained from the cell line ECACC 87042308, if the anti-20 gens present in the serum are fractionated according to their molecular weight by gel filtration chromatography, and the fractions from the chromatography are used in a radioimmunoassay. It emerges from this that the antigen fraction which is also detected on analysis with poly-25 clonal antibodies and has the molecular weight of the breakdown product Col 1 is not detected by the monoclonal antibodies according to the invention. Figure 3 shows the elution profile of the gel filtration chromatography of human serum as shown by the monoclonal antibody, com-30 paring with the elution profile as shown by polyclonal antibodies. Peak 4/4a corresponds to intact procollagen type III or pN collagen type III CRohde H. et al. Eur. J. Biochem. 135, 197 (1983)]. Peak 5/5a corresponds to intact amino-terminal procollagen type III (PIIIP), 35 whereas peak 6/6a corresponds to Col 1 together with breakdown products of amino-terminal procollagen peptide type III with the same molecular weight as Col 1 CRohde H. et al. Eur. J. Biochem 135, 197 (1983)]. The concentration of the substances in the relevant fractions can be determined with III calibration - 5 - the aid of a procollagen peptide type plot.

It is important for the preparation of the antibodies 5 according to the invention that a suitable source is available for obtaining the antigen. As already mentioned, highly purified human or animal procollagen pep- * tide (type III) is advantageously isolated by the process of European Patent 4940, entailing breakdown of 10 tissue or pathological body fluids with collagenase, and removal of the procollagen peptide from the reaction solution and purification by combination of chromatographic methods and/or immunoadsorption. 15 The monoclonal antibody according to the invention can be used in various immunological methods, including all types of radioimmunoassay, for example sequential saturation analysis or equilibrium analysis, where appropriate after labeling with chloramine T or Bo 11on-Hunter 20 reagent CFelber, Meth. Biochem. Anal. 2_2, 1 ( 1974); Shelley et al., Clin. Chem. _1_9, 146 ( 1975)3 and in other competitive and non-competitive binding assays, such as fluorescence or enzyme immunoassays, chemiluminescence or other immunoassays, including immunoradiometric assay 25 (IRMA). The monoclonal antibody can thus be used in immunological methods for the isolation and characterization and for the quantitative determination of procollagen peptide (type III) in tissues and body fluids.

The methods used are known per se to the expert and en-30 tail reacting a liquid sample with contains procollagen peptide (type III) with the monoclonal antibody according to the invention, either in solution or on a solid carrier, and determination of the amount of procollagen peptide (type III) via the antigen/antibody complex for-35 med. It is of no consequence for this whether the procollagen peptide (type III) is still linked to the amino terminus of procollagen (type III) or not. The breakdown products of procollagen peptide (type III), especially Col 1, which have hitherto interfered in the - 6 - immunological determination, are not among the species detected by the antibody according to the invention.

The invention is explained further in the examples which 5 follow. Unless otherwise indicated, percentage data relate to weight.

Example 1 10 Preparation of procollagen peptide (type III) (P III P): Procollagen peptide (type III) is prepared by exposure of procollagen (type III) to collagenase at 37°C. The peptide is not exposed to any denaturing agents during 15 this. A modified process is employed for the preparation of larger amounts of the peptide. All the steps in the process up to the exposure to collagenase are carried out in a cold room. The various NaCl solutions used for solubilization contain 0.05 M Tris-HCl, pH 7.A, 0.01 M 20 EDTA, sodium azide (200 mg/ml) and the protease inhibitors phenyImethyIsu I fony I fluoride (3 pg/ml) and p-chloro-mercuribenzoate (3 yg/ml).

Fetal calf skin (3 kg) is homogenized and extracted for 25 two days in 10 I 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 stirring overnight, the precipitate is collected by cen-trifugation (1800 x g, 20 minutes), washed twice with 30 2.5 M NaCl and redissolved by stirring overnight in 10 I of 0.5 M 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 35 suspended in 2 I of 0.05 M Tris-HCl (pH 8.0) and after addition of 0.02 M CaCl2 the mixture is heated at 50°C for 20 minutes and then incubated together with 1500 U of bacterial collagenase (CLSPA, Worthington, USA) per gram of wet precipitate at 37°C for 3 hours. After the - 7 - exposure to collagenase, the precipitate which is formed is removed by centrifugation, and the solution is dia-lyzed against 0.005 M Tris-HCl, pH 8, 6.8 M urea and passed through a DEAE-cel lulose column (5.0 x 30 cm) 5 which has been equilibrated with the same buffer.

The proteins bound to the column are washed out with NaCl t solutions with the concentration thereof rising from 0 to 0.3 M. The total amount eluted is 2 I. The solution 10 flowing out of the column is examined for the absorption at 236 nm and its antigen activity by use of antibodies which are specific for the amino-terminal segment of procollagen (type III). It is normally the last peak eluted from the column which contains the procol-15 lagen peptide (type III). Salts are removed from the peptide by dialysis against distilled water, and it is freeze-dried. Subsequent purification is carried out on a column of agarose A 1.5 M (2 x 120 cm) which is equilibrated with 1 M CaCl2, 0.05 M Tris-HCl, pH 7.5. 20 Example 2 Hybridoma production 25 Mice of the SJL strain are immunized intramuscularly with 5 yg of procollagen peptide (type III) which has been obtained as in Example 1, in the presence of complete Freund's adjuvant. The immune response is boosted after 4 weeks and after three months by a further intra-30 muscular injection of 5 yg of procollagen peptide (type III) in the presence of incomplete Freund's adjuvant.

Three days before the fusion, the immune response is boosted by intraperitoneal injection of a further 50 yg of procollagen peptide (type III). 35 For the fusion, the animals are sacrificed, and the spleen cells are isolated. The spleen cells are fused with the myeloma cell line P3X63AG8.653 in the presence of polyethylene glycol. The fusion mixture is cultivated - 8 - in hypoxanthine/aminopterin/thymidine medium for a period of two weeks to select for spleen cell x P3X63AG8.653 hybrids. The resulting cell clones are subcloned several times to attain a stable cell line. The resulting 5 cell clones are assayed for antibody production in a radioimmunoIogica I binding assay. The resulting cell line, from which the monoclonal antibody P III P 296 is obtained, is deposited at ECACC under the number 87042308. 10 Example 3 RadioimmunoIogica I binding assay 300 yl of cell culture supernatant, or another sample 15 such as, for example, ascites after cultivation of cells in the abdominal cavity of mice, are incubated with 12 5 100 ul of a I-procoI I agen peptide (type III) solution (1 ng of protein/100 yl prepared as described in European Patent 4940, Example 1) overnight. The antigen/anti-20 body complexes which are formed are precipitated by addition of anti-mouse IgG serum from sheep or another species. After centrifugation and decantation of the supernatant, the amount of precipitated radioactivity is determined in a y~scintillation spectrometer. 25 Example 4 Radioimmunoassay 30 0.2 ml of the sample which is to be analyzed, or of procollagen peptide (type III) standard, is incubated at 4°C overnight with an amount of P III P 296 (in 0.1 ml of buffer) which is limiting with respect to the amount of 12 5 labeled antigen, and 0.1 ml of I-labeled procollagen 35 peptide (type III) (contains 1 ng of protein). The mixture is then incubated with a previously tested amount of anti-mouse IgG serum from sheep in the presence of 10% polyethylene glycol (PEG 6000) for 1 h. The precipitated antigen/antibody complexes are spun down (1500 x g) - 9 - and, after decantat ion, the radioactivity is determined in a y-s c i n t i •-L a t i on spectrometer.

It is then possible, by comparison with a calibration 5 plot which has been constructed by using standards with different amounts of procollagen peptide (type III), to determine the concentration of procollagen peptide (type III) in the unknown solution. Figure 1 shows the calibration plot (1) and serum dilution plots (2) with PIIIP 10 296 (a) comparing with the method of European Patent 4940 using polyclonal antibodies (b).

Example 5 15 RadiolabeIing of the P III P 296 0.2 ml of a solution containing 0.2 mg of monoclonal P III P 296 in 0.05 M phosphate buffer, pH 7.4, is placed in a polystyrene assay tube (12 x 55 mm), and 100 MBq of 20 Na^^I solution, buffered with 10 pi of 0.5 M phosphate buffer, pH 7.4, are added. Addition of 50 ul of an aqueous solution of 20 pg of chloramine T is followed by mixing for 1 min. The iodination reaction is then stopped by addition of 50 yl of an aqueous solution of 25 20 yg of sodium disulfite.

The unreacted Na^^I is then removed from the ^^1-labeled P III P 296 by chromatography on an anion exchanger. The chromatographic fractions which contain 125 20 the purified I-labeled antibody are diluted with a solution of 20 g of Tween 20 and 14.6 g of NajEDTA in one liter of 0.05 M Tris-HCl buffer, pH 8.0, so that the concentration of the labeled antibody is 200 ug/l. 25 Example 6 Antibody coating of assay tubes To immobilize the antibodies on polystyrene assay tubes - 10 - (12 x 75 mm), 300 yl of a solution of 4 mg of monoclonal P III P 296 per liter of 0.01 M 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. Then 500 yl of a 1% strength solution of bovine serum albumin in 0.05 M Tris-citrate buffer, pH 7.5, are added to each tube and incubated at room temperature overnight. The solution is then removed by aspiration. The antibody-coated tubes are dried over silica gel.

Example 7 Immunoradiometric assay 0.1 ml of the sample which is to be analyzed, or 0.1 ml of procollagen (type III) standard, is incubated in polystyrene assay tubes which have previously been coated with 1.2 yg of the monoclonal P III P 296, with the addition of 0.1 ml of phosphate-buffered saline (PBS), at room temperature for 2 hours. The assay tubes are then washed twice with 1 ml of PBS, with decantation, each time. 1 7 S Then 200 yl of I-labeled monoclonal P III P antibodies (contains 40 ng of antibodies) are placed in the tubes and incubated at room temperature for 2 hours. The anti-12 5 body/antigen/ I-antibody complexes bound to the assay tube wall are washed twice with 1 ml of PBS, followed by decantation, each time, and their radioactivity is determined in a scintillation counter.

It is then possible, by comparison with a calibration plot which has been constructed by using standards with different amounts of procollagen peptide (type III), to calculate the concentration of procollagen peptide (type III) in the unknown sample solution. Fig. 2 shows the calibration plot of the radioimmunometric assay. (B/T denotes: bound/total antibody).

Example 8 - 11 - Determination of the molecular weight distribution of the antigens which react with P III P 296 and are present in human serum. 1 ml of serum is fractionated by gel filtration chromatography on an allyldextran crosslinked with N,N'-methy-lenebisacrylamide Pi ephacryl S 300 column (1.6 x 130 cm)] equilibrated in phosphate-buffered saline (PBS) containing 0.04% of a non-ionic detergent, for example a po I yoxyethyIene sorbitan monolaurate (Tween 20) in 3.3 ml fractions. 0.2 ml of each of the fractions is used in the radioimmunoassay described in Example 4. Figure 3 shows the elution profile of the antigen determined using PIIIP 296, comparing with the profile of the antigen determined using the polyclonal antibodies: Peak 4/4a corresponds to pN collagen and intact amino- terminal procollagen (type III) Peak 5/5a corresponds to amino-terminal procollagen peptide (type 111) = (P III P) Peak 6/6a corresponds to Col 1 and breakdown products of amino-terminal procollagen peptide (type III) with the same molecular weight as Col 1.

The concentration of the substances in the relevant fractions can be determined using a procollagen peptide (type III) calibration plot.

Claims (17)

- 12 - HOE 87/F 131 Patent claims:

1. A monoclonal antibody having the reaction pattern which is depicted in Figure 3 towards intact procollagen (type III) and pN collagen (procollagen which is lacking the C-terminal propeptide, peak 4a), intact amino-terminal procollagen peptide (type III) (peak 5a), and Col 1 and breakdown products of the amino-terminal procollagen peptide (type III) having the same molecular weight as Col 1 (peak 6a).

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 or 2, which is assigned to the class IgG.

4. A monoclonal antibody as claimed in one or more of claims 1 to 3, which is formed by a hybridoma which is produced by fusion of cells of a myeloma line and of lymphocytes from an animal which has previously been immunized with procollagen peptide type III.

5. A monoclonal antibody PIIIP 296 of the hybridoma cell line ECACC 87042308.

6. A hybridoma cell line which produces an antibody as claimed in claims 1 to 5 and is formed by fusion of cells of a myeloma cell line and of lymphocytes from an animal which has previously been immunized with procollagen pept ide (type III).

7. The hybridoma cell line ECACC 87042308.

8. A process for the preparation of a against amino-terminal procollagen which comprises monoclonal antibody pept ide (type III), - 13 - a) immunization of animals with amino-terminal procollagen peptide (type III), b) isolation of lymphocytes and fusion thereof with myeloma cells, c) selection of the hybrids for the presence of an antibody having the properties indicated in one of claims 1 to 5, and cloning thereof, and d) isolation of the antibodies from the said clones.

9. The process as claimed in claim 8, wherein the hybridoma cell line ECACC 87042308 is used for carrying out step d) .

10. A method for the quantitative immunological determination of procollagen peptide (type III) using antibodies, which comprises a) reacting a liquid sample which contains amino-terminal procollagen peptide (type III) or procollagen (type III) with a monoclonal antibody as claimed in one or more of claims 1 to 5, and b) determining the amount of amino-terminal procollagen peptide (type III) or the procollagen via the antigen/ antibody complex which is formed.

11. 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 one or more of claims 1 to 5, mixed with a diagnosticalLy acceptable vehicle.

12. A monoclonal antibody according to claim 1, substantially as hereinbefore described and exemplified.

13. A hybridoma cell line according to claim 6, substantially as hereinbefore described and exemplified.

14. A process according to claim 8 for the preparation of a monoclonal antibody, substantially as hereinbefore described and exemplified.

15. A monoclonal antibody whenever prepared by a process claimed In any one of claims 8, 9 or 14.

16. A method according to claim 10 for the quantitative immunological determination of procollagen peptide (type 111) using antibodies, substantially as hereinbefore described and exemplified.

17. A diagnostic composition according to claim 11, substantially as hereinbefore described. Dated this the 29th day of April, 1988 F. R. KELLY & CO. 27 Cly . „ . Dublin 4 AGENTS FOR THE APPLICANTS. BY: EXECUTIVE