DK169571B1 - Monoclonal antibody, method for its preparation, hybridoma cell line producing the antibody, methods for quantitative immunological determination of procollagen peptide (type III) with the antibody, and diagnostic composition containing the monoclonal antibody - Google Patents

Abstract

The said peptide can be determined with great accuracy with the aid of a monoclonal antibody which is directed specifically against one 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).

Description

in DK 169571 B1

The present invention relates to a monoclonal antibody, to a method for its preparation, to a hybridoma cell line producing the antibody, to quantitative immunological determination of procollagen peptide 5 (type III) with the antibody, and to a diagnostic composition containing the monoclonal antibody. .

Procollagen peptide (type III) (P III P) is the amino-terminal propeptide in collagen (type III) that is cleaved extracellularly after secretion of the procollagen (type III) -10 molecule. Procollagen peptide (type III) can, for its part, be cleaved to collagen fragments col I, col 2 and col 3, which can be isolated by known protein chemical methods (Nowack, H. et al., Eur .

J. Biochem. 20 / 205-216 (1976), Bruckner, P. et al., Eur. J.

Biochem. 90, 595-603 (1978)).

By a radioimmunological assay method as described in European Patent No. 4940, the concentration of this procollagen peptide in body fluids can be determined. Knowledge of the serum concentration of the peptide 20 enables statements about the activity of fibrotic disease states, e.g. in the liver (Rohde, H. et al., Eur. J. Clin. Invest.

9, 451-459 (1979)).

An exact, selective determination of procollagen peptide (type III) and procollagen (type III) in serum and other body fluids is proposed in European Patent Application No. 289,930. However, a number of centrifugation steps are necessary, which make it difficult to apply this study in the routine laboratory. Simpler handling enables the "Immunoradiometric Assay" (IRMA) test method. In this method, two antibodies are used, one of which is coupled to a solid support, thereby eliminating a precipitation step and thus a centrifugation and some pipetting step.

Surprisingly, a monoclonal antibody has now been invented that has a specific effect on an intact 35 procollagen (type III) epitope that has not hitherto been recognized as a particular peak by gel filtration chromatography. In combination with other monoclonal or polyclonal antibodies with specificity for procollagen peptide (type III) and / or procollagen (type III), this antibody allows the determination of these antigens by IRMA technique.

This invention relates to a monoclonal antibody having specific action against an epitope on intact procollagen (type III), pN collagen, intact amino-terminal procollagen peptide (type III), and degradation products of the 10 amino-terminal procollagen peptide (type III) which, in terms of molecular weight, lies between the amino-terminal procollagen peptide (type III) and col 1's, and exhibits a weak reaction against col 1 and degradation products of the amino-terminal procolagen peptide (type III) having the same molecular weight as col 1 .

A hybridoma cell line that produces an antibody of the invention formed by fusion of cells from a myeloma cell line and from lymphocytes of a pre-collagen peptide (type III) animal.

A method of producing a monoclonal antibody of the invention in which a) animals are immunized with amino-terminal procollagen peptide (type 25 III), b) lymphocytes are recovered and fused with myeloma cells, c) the hybrids are selected for the presence of an antibody having the above characteristics and clones; and d) the antibody is recovered from said clones.

A method for quantitative immunological determination of procollagen peptide (type III) with an antibody of the invention, wherein: a) a liquid sample containing amino-terminal procollagen peptide (type III) or procollagen DK 169571 B1 3 (type III) ) are reacted with the monoclonal antibody of the invention, and b) the amount of the amino-terminal procollagen peptide (type III) or of the procollagen (type III) is determined over the antigen-antibody complex formed.

A method for quantitative immunological determination of procollagen peptide (type III) with an antibody of the invention, wherein a) the monoclonal antibody of the invention is coupled to a solid matrix, b) a liquid sample containing amino-terminal procollagen peptide or procollagen is reacted with said antibody, and c) the bound antigen is detected with labeled monoclonal or polyclonal antibodies with specificity for procollagen peptide (type III) or procollagen (type III).

6. A diagnostic composition for determining the amount of procollagen peptide (type III) in body fluids, 20 and compositions of the invention is characterized in that it contains an effective amount of a monoclonal antibody of the invention alone or in combination with other antibodies in admixture. with a diagnostically acceptable carrier. In particular, as with other antibodies, an effective amount of the monoclonal antibody proposed in DK Patent Application No. 2328/88 is used.

In the following, the invention will be described in detail, in particular the preferred embodiments, with reference to the accompanying drawings, in which: Figure 1 shows the calibration curve for the immunodiometric analysis performed in Example 6; 2 shows the reaction pattern for the monoclonal antibody P-III-P 226 and for a polyclonal antibody; and FIG. 3 shows the reaction pattern for the monoclonal antibody P-III-P 296 and for a polyclonal antibody.

4 DK 169571 B1

To produce the monoclonal antibodies, animals, preferably rodents, e.g. mice, rats, rabbits and guinea pigs are immunized with procollagen peptide (type III) isolated by the method of European patent no.

5 4940, in the presence of adjuvant. It is particularly preferred to use mice, especially those from the SJL strain. With repeated secondary injections, e.g. at 4-8 weeks distance the immune response is enhanced. The result of immunization is checked by determining the concentration of antibodies in the 10 radiological binding experiment (R. Timpl and L. Risteli, Immunochemistry of the extracellular matrix, H. Forthmayr, vol. 1, 199 (1982)). A few days before the fusion of the lymphocytes with a myeloma cell line, the animals are treated with pro-collagen peptide (type III) without adjuvant. The lymphocytes of the animals are recovered and fused with a myeloma cell line, which may also be derived from one of the above animal species, preferably from mice, especially with the cell line P3X63AG8,653. Advantageously, lymphocytes are fused with myeloma cell lines of the same species. The fusion and further culturing of the cell clones 20 is carried out in a manner known to those skilled in the art, with the concentration of specific antibodies in the cell culture over-phase being determined by immunological binding assays, from the cell clones shown in this assay, a clone is selected for use in IRMA. It is particularly preferred to work with a cell line produced by fusion of lymphocytes from SJL strain immunized mice of the SJL strain with the mouse myeloma cell line P3X63AG8,653 and produce a monoclonal antibody with the one shown in FIG. 2. This cell line was deposited on March 2, 1988, under the terms of the Budapest Treaty with European

Collection of Animal Cell Cultures (ECACC), PHLS Center for Applied Microbiology and Research, Porton Down, Salisbury, SP40JG, United Kingdom with the number 88030202.

The monoclonal antibodies of the invention belong to the class of IgG, IgM and IgA proteins. Antibodies of the class IgG, especially of the subclass IgG2b, are used with particular benefit to DK 169571 B1. The properties of the monoclonal antibodies are elucidated by the monoclonal antibody PIIIP 226 recovered from the cell line of ECACC 88030202 when the antigens present in the serum are separated by gel filtration chromatography according to their molecular weight and the chromatographic fractions are used in radioimmunoassay. In FIG. Figure 2 shows the elution profile of the gel filtration chromatography showing the monoclonal antibody PIIIP 226 in comparison with the elution profile exhibited by the polyclonal antibodies. Tip 1 / la corresponds to 10 intact procollagen (type III) and pN collagen (type III), respectively. Point 2 / 2a corresponds to intact amino-terminal procol layer peptide (type III) and its degradation products of similar size. Point 3a comprises degradation products of procollagen peptide (type III), which gives a smaller peak 15 in the chromatogram than procollagen peptide (type III), but clearly larger than col 1. Thus, in each case, the molecular weight of the degradation products is between ca. 45,000 (procollagen peptide) and approx. 10,000 (col 1). Point 4 / 4a corresponds to col 1 and degradation products of the amino-terminal procol layer 20 (type III) peptide of the same molecular weight as col 1. It appears that the antigen fraction included in the analysis with polyclonal antibodies having molecular weight which is of the degradation product col 1, or is col 1, is clearly included weaker by the monoclonal antibody of the invention. Thus, the monoclonal antibody of the invention has a specific effect against an epitope on the amino-terminal procollagen peptide (type III) that is not present in col 1. Furthermore, some of the antigens included in tip 2 are recognized by polyclonal antibodies. , not by antibody PIIIP 226, so that at the position of tip 2, two peaks (2a and 3a) are recognized with the antibody PIIIP 226. Point 3a is also not found with the antibody PIIIP 296 proposed in the German patent application.

For the preparation of the antibodies of the invention 35, it is important that an appropriate source of recovery of the antigen is provided. As already mentioned, human DK 169571 B1 g or animal highly purified procollagen peptide (type III) is advantageously isolated by the method of European Patent No. 4940, wherein tissue or pathological body fluids are digested with collagenase and the procollagen peptide is separated from the reaction solution. and purified by combination of chromatographic methods and / or immunosorption.

The monoclonal antibodies of the invention can be used in various immunological methods, including all forms of radioimmunoassay, e.g. sequential saturation or equilibrium analysis, and in other competing and non-competing binding assays, such as fluorescence, enzyme, chemiluminescence or other immunoassays.

It is particularly suitable for use in immunosorbent assays in the sandwich method. Thus, the monoclonal antibody can be used in immunological methods for isolation and characterization as well as for the quantitative determination of procollagen peptide (type III) in tissues and body fluids. One skilled in the art will proceed according to methods known per se, advantageously by reacting a liquid assay containing procollagen peptide (type III) with the monoclonal antibody of the invention coupled to a solid matrix, preferably composed of plastic material, especially small plastic glass, and the amount of procollagen peptide (type III) is determined by binding of polyclone or other monoclonal antibody, preferably the monoclonal antibody proposed in European Patent Application No. 289,930. is provided with a radioactive or other marking. This method can also be carried out by binding polyclonal or monoclonal antibodies, especially the monoclonal antibody 30 proposed in DK patent application 2323/88, to a solid matrix and reacting with the antigen, thereby quantitating this antigen by binding the labeled antibody of the invention. In these methods, it does not matter whether or not the procollagen peptide (type III) is bound to the amino terminus of the procollagen (type III). The antibody disrupting degradation products of procol layer peptide (type III), in particular col I, are not included in the assay system described so far by the immunological assay by polyclone DK 169571 B1 7.

The 5 monoclonal antibody proposed in European Patent Application No. 289,930 exhibits that shown in FIG. 3 depicted reaction pattern to intact procollagen (type III) and pN collagen (procollagen lacking the C-terminal propeptide, tip 4a), intact amino-terminal procollagen peptide (type III) (tip 5a), and col 1 and degradation products thereof amino-terminal procollagen peptide (type III) having the same molecular weight as col 1 (tip 6a).

To prepare this monoclonal antibody, proceed essentially as described above. It is particularly preferred to work with a cell line produced by fusion of lymphocytes from mice of the SJL strain immunized with procollagen peptide (type III) with the mouse myeloma cell line P3X63AG8,653. This cell line is deposited with the European Collection of Animal Cell Cultures (ECACC), PHLS Center for Applied Microbiology and Research, Porton Down, Salisbury, 20 SP40JG, United Kingdom, under landfill number 87042308.

In the following examples, the invention is further explained. Percentages refer to weight unless otherwise stated.

Example 1 Preparation of procollagen peptide (type III)

Procollagen peptide (type III) is produced by the action of collagenase on procollagen (type III) at 37 ° C. This does not expose the peptide to any denaturing agents. For the production of larger amounts of the peptide, a modified process is used. All process steps are carried out in cold storage until The various sodium chloride solutions used for solubilization contain 0.05 M tris * HCl, pH 7.4, 0.01 M EDTA, 200 mg / ml sodium azide as well as 3 μg / ml phenylmethylsulfonyl fluoride and 3 µg / ml β-chloromercuribenzoate as protease inhibitors.

3 kg of fetal calf skin is homogenized in 10 liters of 1 M

δ

NaCl and extracted for 2 days. Dissolved collagen is precipitated from the extraction solution by the addition of solid sodium chloride to a final concentration of 2.5 M. After stirring overnight, the precipitate is collected by centrifugation (1,800 xg, 5 minutes), washed twice with 2.5 M NaCl and dissolved again. it is stirred overnight in 10 liters of 0.5 M MaCl. Small amounts of insoluble material are removed by centrifugation. The mixture of collagen (type III) and procollagen (type III) thus obtained is precipitated with 1.6 M NaCl. The precipitate is then suspended in 2 liters of 0.05 M tris * HCl (pH 8.0) and heated after addition of 0.02 M CaCl 2 for 20 minutes to 50 ° C and then incubated for 3 hours at 37 ° C together with 1,500 U bacterial collagenase (CLSPA, Worthington, USA) per grams of damp precipitate. Following the action of the collagenase, the precipitate formed is separated by centrifugation and the solution is dialyzed against 0.005 M tris * HCl, pH 8.0, 6.8 M urea and applied to a DEAE cellulose column (5.0 x 30 cm) which is equilibrated by the same buffer.

The proteins bound to the column are washed out with a sodium-chloride solution, the concentration of which increases from 0 to 0.3 M. The total elution amounts to 2 liters. The column flowing solution is examined for the absorption at 236 nm and the antigenic activity using antibodies specific for the amino-terminal segment of the procollagen (type III). Usually, the last tip eluted from the column contains the procollagen peptide (type III). The peptide is desalted by dialysis against distilled water and lyophilized. The further purification takes place on a column with agarose A 1.5 M (2 x 120 cm) (Fa. Biorad) equilibrated with 1 M CaCl 2, 0.05 M tris ^ HCl, pH 7.5.

Example 2

Hvbridomfremstilling

Mice of the SJL strain are immunized intramuscularly with 5 35 µg procollagen peptide (type III) obtained according to Example 1, in the presence of complete Freund's adjuvant. After 4 weeks, and after 3 months, the immune response is enhanced by a further intramuscular injection of 5 μg procollagen peptide (type III) in the presence of incomplete Freund's adjuvant. Three days before the fusion, the immune response is enhanced by the injection of an additional 50 µg procollagen peptide (type III).

For fusion, the animals are killed and the spleen cells are isolated.

In the presence of polyethylene glycol, the spleen cells are fused with the myeloma cell line P3X63AG8,653. (The resulting cell line is deposited with ECACC under number 88030202.) 10 When culturing the fusion mixture in hypoxanthine aminopterin - thymidine medium over a period of 2 weeks, select for spleen cell x P3X63AG8,653 hybrids. To obtain a monoclonal cell line, the cell clones obtained are subcloned several times. The upper phases of the cell colonies formed are examined for antibody production by radioimmunological binding experiments. Thus, the bimonoclonal antibody PIIIP 226 is obtained.

Example 2

Radioimmune binding experiment 20 300 μΐ cell culture superphase or another sample, e.g. ascites after culturing hybridoma cells in the abdominal cavity of mice are incubated overnight at 100 μΐ of a solution of 12 µg procollagen peptide (type III) (1 ng protein / 100 μΐ, prepared as in European Patent No. 4940, Example 25 1 ). The antigen-antibody complexes formed are precipitated by the addition of anti-rous IgG serum from sheep or another species. After centrifugation and decantation of the upper phase, the amount of precipitated radioactivity is determined in the τ scintillation spectrometer.

30

Example 4

Radioactive labeling of the antibodies 0.2 ml of solution with 0.2 mg of the monoclonal antibody PIIIP 296 recovered according to German patent application P 37 14 633.5, Example 2 or another antibody in 0.05 M phosphate buffer, pH 4, placed in a small polystyrene test tube 10 DK 169571 B1

(12 x 55 mm) and 100 MBq of Na125J solution are added, buffered with 10 μΐ 0.5 M phosphate buffer, pH 7.4. After adding 50 μΐ aqueous solution of 20 µg chloramine T, mix for 1 minute. Then, the iodination reaction is terminated by the addition of 50 μΐ aqueous solution of 20 μg sodium disulphite. The unreacted Na125J is then separated from the 125J-labeled antibody by chromatography on an anion exchanger. The chromatographic fractions containing the purified 125 J-labeled antibody are diluted with a solution of 20 g of Tween 20 and 14.6 g of Na 2 EDTA in 1 liter of 0.05 M tris * HCl, pH

8.0 so that the concentration of the labeled antibody is 200 μg / liter.

Example 5 Coating of test tubes with antibodies

To fix antibodies to small polystyrene test tubes (12 x 75 mm), apply 3 oo μ oo solution of 4 mg / liter antibody to each glass, e.g. PIIIP 226, in 0.01 M sodium phosphate buffer, pH 6.4, and incubate overnight at room temperature. Subsequently, the antibody solution is aspirated and 500 μΐ 1% solution of bovine serum albumin in 0.05 M tris · citrate, pH 7.5, is added to each glass. After overnight incubation at room temperature, the solution is aspirated. The antibody-coated glasses are dried over silica gel.

25

Example 6

Immunodiometric Assay (Radioimmunometric Assav. IRMA) 0.1 ml of the sample to be assayed or of the procollagen peptide (type III) standard is incubated for 2 hours at room temperature in polystyrene test tubes coated with Example 5 monoclonal antibody PIIIP 226 with the addition of 0.1 ml of phosphate buffered saline (PBS). Then the tubes are washed twice with 1 ml of PBS each. Then, 35 µl of 125 J-labeled antibody PIIIP 296 (= 40 ng antibody) or another antibody is placed and incubated for 2 hours at room temperature. The radioactivity of the antibody-antigen-L 2 µ J antibody complexes bound to the glass wall is determined in -r scintillation spectrometer after two washings with 1 ml of PBS each and subsequent decantation.

By comparing a calibration curve for which standards with different amounts of procollagen peptide (type III) have been used, the concentration of procollagen peptide (type III) in the unknown sample solution can be calculated. FIG. Figure 1 shows the calibration curve for the immuno-radiometric analysis (B / T means the ratio of bound to total radioactivity used).

Example 7

Determination of the Molecular Weight Distribution of the Human Serum Antibody Reagents with 15 PIIIP 226 1 ml serum is separated by gel filtration chromatography on an allyl dextran branched with Ν, Ν'-methylene bisacrylamide [Sephacryl S 300 column (1.6 x 130 cm)] with 0.4% nonionic detergent, e.g. a polyoxyethylated sorbitan monolaurate (Tween 20). 0.2 ml of each fraction is incubated overnight at 4 ° C with a proportionate amount of the antibody to be examined (in 0.1 ml buffer) and 0.1 ml 125 J labeled procollagen peptide (type III) (contains 1 ng of protein). Then incubate for 1 hour with a previously tested amount of sheep anti-mouse IgG serum in the presence of 10% polyethylene glycol (PEG 6,000). The precipitated antigen-antibody complexes are centrifuged (1,500 x g) and, after decantation, the radioactivity is determined in -τ scintillation spectrometers. By comparison with a calibration curve for which standards with different amounts of procollagen peptide (type III) have been used, the concentration of the antigens reacting with the antibody used in the chromatography fractions can be determined. FIG. 2 shows the elution profile 35 of the antigen determined by PIIIP 226 in comparison with the profile of the antigen determined by polyclonal antibodies.

Claims (16)

1. Monoclonal antibody, characterized in that it has specific action against an epitope on intact procollagen (type III), pN collagen, intact amino-terminal procollagen peptide (type III), and degradation products of the amino-terminal procollagen peptide (type III) which lies in molecular weight between the amino-terminal procollagen peptide (type III) and col 1's, and exhibits a weak reaction 10 to col 1 and degradation products of the amino-terminal procollagen peptide (type III) of the same molecular weight as col 1. The monoclonal antibody of claim 1 having specific action against an epitope on the amino-terminal procollagen-15 peptide (type III) not present in col 1. Monoclonal antibody according to claim 1 or 2, characterized in that it belongs to the class of IgG. Monoclonal antibody according to one or more of claims 1 to 3, characterized in that it is formed by a hybridoma that is formed by fusion of cells of a myeloma line with lymphocytes from a pre-immuno-terminal procollagen peptide (type III) immunized animals. 25 III) are immunized, b) lymphocytes are recovered and fused with myeloma cells, c) the hybrids are selected for the presence of an antibody having the properties set forth in any of claims 1-3 and cloned, and d) the antibody is recovered from the aforementioned clones. 5. Monoclonal antibody PIIIP 226 from the hybridoma cell line ECACC 88030202. A hybridoma cell line producing an antibody 30 according to claims 1-5, formed by fusion of cells from a myeloma cell line and from lymphocytes of a pre-collagen peptide (type III) animal. 7. The hybridoma cell line ECACC 88030202. DK 169571 B1 A method of producing a monoclonal antibody according to one or more of claims 1-5, characterized in that a) animals with amino-terminal procollagen peptide (type The method according to claim 8, characterized in that the hybridoma cell line ECACC 88030202 is used to perform step d). Method for quantitative immunological determination of procollagen peptide (type III) with antibodies, characterized in that a liquid sample containing amino-terminal procollagen peptide (type III) or procollagen (type III) is reacted with the monoclonal antibody according to one or more of claims 1-5, and b) the amount of the amino-terminal procollagen peptide 25 (type III) or of the procollagen (type III) is determined over the formed antigen-antibody complex. A method for quantitative immunological determination of procollagen peptide (type III) with antibodies, characterized in that a) the monoclonal antibody according to one or more of claims 1-4 is coupled to a solid matrix, b) a liquid sample which containing amino-terminal procollagen peptide or procollagen, is reacted with said antibody, and c) the bound antigen is detected with marked monoclonal DK 169571 B1 or polyclonal antibodies with specificity for procollagen peptide (type III) or procollagen (type III). Method according to claim 11, characterized in that in step a), polyclonal or monoclonal antibodies with specificity for procollagen peptide (type III) and / or procollagen (type III) are used and in step c) a monoclonal antibody according to a or more of claims 1-5, 10 wherein the antibody is labeled. The method according to claim 11, characterized in that in step a), as a monoclonal antibody, an antibody exhibiting specific action against an epitope 15 of intact procollagen (type III), pN collagen and intact amino-terminal procollagen peptide (type) is used. III) and a weak reaction against col 1 and degradation products of the amino-terminal procollagen peptide (type III) of the same molecular weight as col l. Method according to claim 12, characterized in that in step a), as a monoclonal antibody, an antibody is shown which exhibits specific action against an epitope of intact procollagen (type III), pN collagen (tip 4a) and intact amino-terminal procollagen. peptide (type III) (tip 5a) and a weak reaction to col 1 and degradation products of the amino-terminal procollagen peptide (type III) of the same molecular weight as col 1 (tip 6a). The method of claim 13 or 14, characterized in that, as a monoclonal antibody, PIIIP 296 is used from the hybridoma cell line ECACC 87042308. Diagnostic composition for determining the amount of procolagen peptide (type III) in body fluids, characterized by an effective amount of the monoclonal anti DK 169571 B1 substance according to one or more of claims 1-5 alone or in combination with others. antibodies in admixture with a diagnostically acceptable carrier.