NO173104B - MONOCLONAL ANTIBODY FOR SELECTIVE IMMUNOLOGICAL DETERMINATION OF INTACT PROCOLLAGEN Peptide (TYPE III) AND PROCOLLAGEN (TYPE III), A HYBRIDOMA CELL LINE PRODUCING THIS ANTIBODY AND A PREPARED CONTENT - 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

The present invention relates to a monoclonal antibody of the class IgG for use in the selective immunological determination of intact pro-collagen peptide (type III) and pro-collagen (type III) in body fluids.

The invention also relates to a hybridoma cell line that produces such an antibody.

Finally, the invention relates to a diagnostic preparation for determining the amount of procollagen peptide (type III) in body fluids.

Procollagen peptide (type III) is the amino-terminal propeptide of collagen (type III), which after cleavage of the procollagen (type III) molecule is cleaved extracellularly. With a radioimmunological determination method, as described in EP-PS No. 4940, the concentration of this procollagen peptide can be determined in body fluids. The knowledge of the peptide's serum concentration enables statements about the activity of fibrotic diseases, for example of the liver [Rohde, H. et al. "Eur. J. Clin. Invest", 9, 451-459 (1979)].

However, the precise, selective determination of procollagen peptide (type III) in serum and other body fluids is not possible with the methods discussed so far, as the polyclonal antibodies used in these methods react with different antigens present in serum, which are partly breakdown products of the procollagen peptide (type III) with different lower affinity [Niemelå, 0. et al. "Clin. Chim. Acta" 124, 39-44 (1982)]. This causes the serum dilution curves and the dilution curves of other body fluids not to parallel calibration curves formed with pure procollagen peptide (type III) and therefore must be determined from any unknown sample of the antigen content in multiple dilutions to determine the antigen concentration above the 50# section of the dilution curve.

With the help of the method in EP-PS 0 089 008, it is possible to solve this technical problem, as antibodies are used which have comparable affinity for intact procollagen peptide (type III) and its breakdown product Col 1. With this method, intact and degraded procollagen peptide (type III) are determined together, which, however, leads to inaccuracy in the diagnostic statement, as the normal collective and the patient collective can overlap strongly.

Surprisingly, a monoclonal antibody has now been found that does not react with the breakdown products of the procollagen peptide (type III) found in body fluids. With this monoclonal antibody, an immunological determination of the amino-terminal procollagen peptide (type III) has been made possible, where its degradation products are not included, and which is distinguished by the fact that serum dilution curves, the dilution curves of other body fluids and the adjustment curve show complete parallelism.

According to this, the present invention relates to a monoclonal antibody of the class IgG, and this antibody is characterized by the ability to react specifically with intact procollagen (type III), pN-collagen, ingested amino-terminal procollagen peptide (type III), however not binding to Col 1 and the degradation products of the amino-terminal procollagen peptide (type III) with the same molecular weight as Col 1.

In a preferred embodiment of the invention, the monoclonal antibody PHI 296 is produced by the hybridoma cell line ECÅCC 87042308.

As mentioned at the outset, the invention also relates to a hybridoma cell line, namely the hybridoma cell line ECACC 87042308, which produces the above-mentioned antibody, and this cell line is characterized by the fact that the cell line is the hybridoma ECACC 87042308 and by the fact that it is formed by the fusion of cells from a myeloma cell line and of lymphocytes of an animal previously immunized with procollagen peptide (type III).

Finally, the invention relates to a diagnostic preparation for determining the amount of procollagen peptide (type III) in body fluids, and this preparation is characterized by the fact that it contains the monoclonal antibody as described above together with a diagnostically acceptable carrier.

The invention is explained in detail below, particularly the preferred embodiments.

To produce the monoclonal antibody, animals, preferably rodents, such as mice, rats, rabbits, guinea pigs, can be immunized with procollagen peptide (type III), which was isolated according to the method in EP-PS 4940 in the presence of adjuvant. Preferably, mice are used, especially those of the SJL strain. With repeated secondary injections, for example at intervals of 4 to 8 weeks, the immune response is enhanced. The result of the immunization is checked by determining the concentration of antibodies in the radioimmunological binding sample (R. Timpl and L. Risteli, "Immunochemistry of the extracellular matrix", H. Furthmayr, Ed., vol. 1, 199

(1982)). Somewhat before the fusion of the lymphocytes with a myeloma cell line, the animals are treated with procollagen peptide (type III) without adjuvant. Lymphocytes of the animals are prepared and fused with a myeloma cell line, which can likewise originate from one of the above-mentioned animal species, preferably, however, from mice, especially with the cell line P3X63AG8.653. Lymphocytes are advantageously merged with myeloma cell lines of the same species. The fusion and the further cultivation of the cell clones is carried out in a manner known to those skilled in the art, the concentration of the specific antibody being determined in the supernatant of the cell culture by means of an immunological binding test. From the cell clones obtained by the fusion, suitable clones are selected for use in immunological methods. Preferably, work is done with a cell line that is produced by fusion of mouse lymphocytes of the SJL strain against procollagen peptide (type III) with the mouse myeloma cell line P3X63ÅG8.653. This cell line is deposited at the European Collection of Animal Cell Cultures (ECACC), PHLS Center for Applied Microbiology and Research, Porton Down, Salisbury, SP40JG, U.K. under number 87042308.

The monoclonal antibodies according to the invention belong to the group of immunoglobulins, preferably to the class of IgG, IgA and IgM proteins. Antibodies of the IgG class, especially of the IgG2B subclass, are particularly useful. The antibody according to the invention is particularly surprising because its affinity to antigens is higher than the affinity of polyclonal antibodies. Usually the opposite is the case. The properties of the monoclonal antibody are clarified with the help of the monoclonal antibody PIIIP 296 produced from cell line ECACC 87042308, when the antigens present in the serum are separated by gel filtration chromatography according to their molecular weight and the fractions of the chromatography are used in radioimmunoanalysis. Thereby it turns out that the antigen fraction which is included in analysis with polyclonal antibodies and has the molecular weight of the breakdown product Col 1 is not covered by the monoclonal antibody according to the invention. In fig. 3 shows the elution profile of gel filtration chromatography of human serum as it shows the monoclonal antibody compared to the elution profile as it has polyclonal antibodies. Peak 4/4a corresponds to intact procollagen (type III) or pN-collagen (type III) respectively [Rohde H. et al. "Eur. J. Biochem." 135, 197 (1983)]. Peak 5/5a corresponds to intact amino-terminal procollagen peptide (type III) (P III P), while peak 6/6a corresponds to Col 1 and degradation products of the amino-terminal procollagen peptide (type III) with the same molecular weight as Col 1 [Rohde H. et al. "Eur. J. Biochem." 135, 197 (1983)]. The concentration of the substances in the corresponding fractions can be determined using a procollagen peptide (type III) adjustment curve.

For the production of the antibodies according to the invention, it is important that a suitable source for the production of the antigen is available. As already mentioned, human or animal, highly purified procollagen peptide (type III) is advantageously isolated by the method according to EP-PS 4940, as tissue or pathological body fluids are broken down with collagenase and the procollagen peptide is separated from the reaction solution and purified by a combination of chromatographic methods and/or immunoadsorption.

The monoclonal antibody according to the invention can be used in various immunological methods, including all forms of radioimmunoanalysis, for example sequential saturation analyzes or equilibrium analyses, optionally after labeling with chloramine T or Bolton-Hunter reagent [Felber, "Meth. Biochem. Anal." 22, 1 (1974), Shelley et al., "Clin. Chem." 19, 146 (1975 )] as well as in other competitive and non-competitive binding assays, such as fluorescence, enzyme, chemiluminescence or other immunoassays, including immunoradiometric assay (IRMA). The monoclonal antibody can therefore be used in immunological methods for isolation and characterization as well as for quantitative determination of procollagen peptide (type III) in tissues and body fluids. One proceeds according to the methods known per se to those skilled in the art, whereby a liquid sample containing procollagen peptide (type III) is reacted with the monoclonal antibody according to the invention, whether it is in solution or on a solid support, and the amount of procollagen peptide ( type III) is determined over the formed antigen-antibody complex. Thereby, it does not matter whether procollagen peptide (type III) is also connected or not with the amino terminus of procollagen (type III). The degradation products of the procollagen peptide (type III), especially Col 1, which have hitherto interfered with the immunological determination, are not covered by the antibody according to the invention.

The invention is explained in more detail in the following examples, where percentages refer to weight, if nothing else is stated.

Example 1

Production of procollagen peptide (type III) (P III P): Procollagen peptide (type III) is produced by the action of collagenase on procollagen (type III) at 37°C. In this way, the peptide is not exposed to any denaturing agents. A modified method is used to produce larger quantities of the peptide. All process steps up to the impact of the collagenase are carried out in a cold room. The various NaCl solutions used for solubilization contain 0.05 M tris-HCl, pH 7.4 0.01 M EDTA, sodium azide (200 mg/ml) and the protease inhibitor phenylmethylsulfonyl fluoride (3 µg/ml) and p-chloromercuribenzoate (3 jjg/nk).

Fetal calf skin (3 kg) is homogenized in 10 1 1 M NaCl and extracted for two days. Dissolved collagen is separated from the extraction solution by adding solid NaCl to a final concentration of 2.5 M. After stirring overnight, the precipitate is collected by centrifugation (1800 x g, 20 minutes), washed twice with 2.5 M NaCl and dissolved again, as it is stirred overnight in 10 1 0.5 M NaCl. Smaller amounts of insoluble material are removed by centrifugation. The thus formed mixture of collagen (type III) and procollagen (type III) is combined with 1.6 M NaCl. The precipitate is then suspended in 2 1 0.5 M tris-HCl (pH 8.0) and heated after the addition of 0.02 M CaCl2 for 20 minutes at 50°C and then incubated for 3 hours at 37°C together with 1500 U of bacterial collagenase (CLSPA, Worthington, USA) per grams of moist precipitate. After the action of the collagenase, the formed precipitate is separated by centrifugation and the solution is dialyzed against 0.005 M tris-HCl, pH 8, 6.8 M urea and passed over a DEAE cellulose column (5.0 x 30 cm), which was equilibrated with the same culture .

The proteins bound to the column are washed out with NaCl solutions, the concentration of which increases from 0 to 0.3 M. The total elution amount is 2 1. The solution draining from the column is examined with regard to the adsorption at 236 nm and its antigenic activity when applied of antibodies specific for the amino-terminal segment of procollagen (type III). Normally, the last peak eluted from the column contains procollagen peptide (type III). The peptide is desalted and lyophilized by dialysis against distilled water. The further purification takes place on a column of agarose A 1.5 M (2 x 120 cm), which is equilibrated with 1 M CaCl£, 0.05 M tris-HCl, pH 7.5.

Example 2

Hybridoma production.

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

For the fusion, the animals are euthanized and the spleen cell is isolated. In the presence of polyethylene glycol, the spleen cells fuse with the Myeloma cell line P3X63AG8.653. When culturing the fusion mixture in the Hypoxanthine-aminopterin-thymidine medium over a period of 2 weeks, spleen cells x P3X63AG8.653 hybrids are selected. To obtain a stable cell line, the formed cell clones are subcloned several times. The formed cell colonies are examined in a radioimmunological binding test for antibody production. The formed cell line, from which the monoclonal antibody P III P 296 is extracted, has been deposited at ECACC under number 87042308.

Example 3

Radioimmune binding assay

300 μl of cell culture supernatant or other sample, for example ascites, after cultivation of cells in the abdominal cavity of mice, is incubated with 100 μl of an I-labeled procollagen peptide (type III) solution, (1 ng protein/100 μl prepared as described in EP-PS 4940, example 1) overnight. The formed antigen-antibody complexes are precipitated by the addition of anti-mouse IgG serum from sheep or another species. After centrifugation and decantation of the supernatant, the amount of the precipitated radioactivity is determined in a γ-scintillation spectrometer.

Example 4

Radioimmune test.

0.2 ml of the sample to be analyzed or the procollagen peptide (type III) standard is incubated overnight at 4°C with a limiting amount of PIIIP 296 (in 0.1 ml buffer) and 0.1 ml 125 J-labelled procollagen peptide (type III) (contains 1 ng protein). It 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 hour. The precipitated antigen-antibody complexes are centrifuged (1500 x g) and, after decantation, the radioactivity is determined in the γ-scintillation spectrometer.

By comparison with one calibration curve, for the preparation of which standards with different amounts of procollagen peptide (type III) were used, the concentration of procollagen peptide (type III) in the unknown solution can then be determined. Figure 1 shows the adjustment curve (1) and the serum dilution curve (2) with PIIIP 296 (a) compared to the method according to EP 4940 with polyclonal antibodies (b).

Example 5

Radioactive marking of P III P 296.

0.2 ml of a solution of 0.2 mg of the monoclonal P III P 296 in 0.05 M phosphate buffer pH 7.4 is placed in a small polystyrene tube (12 x 55 mm) and treated with 100 MBq Na125 J solution, debuffered with 10 µl 0.5 M phosphate buffer f is pH 7.4. After adding 50 ml of an aqueous solution of 20 pg chloramine T, mix for 1 minute. The iodination reaction is then terminated by the addition of 50 µl of an aqueous solution of 20 µg of sodium disulfite.

The unreacted Na^25j is then separated from the 125j_ labeled P III P 296 by chromatography on an anion exchanger. The chromatographic fractions, which contained the purified <125>J-labeled antibody, are diluted with a solution of 20 g of Tween 20 and 14.6 g of Na2EDTA in 1 liter of 0.05 M tris-HCl buffer pH 8, so that the concentration of the labeled antibody was 200 pg/l.

Example 6

Antibody coating of small test tubes.

For fixation of the antibody on small polystyrene test tubes (12 x 75 mm), each tube contains 300 µl of a solution of 4 mg of monoclonal P III P per liter of 0.01 M sodium phosphate buffer f is pH 6.4 and is incubated overnight at room temperature. The antibody solution is then aspirated. 500 µl of a 1 µg solution of bovine serum albumin in 0.05 M tris-citrate buffer pH 7.5 is then placed in each tube and incubated overnight at room temperature. The solution is then aspirated. The antibody-coated tubes are dried over silica gel.

Example 7

Immunoradiometric test.

0.1 ml of the sample to be analyzed or 0.1 ml of procollagen (type III) standard is incubated in small polystyrene test tubes previously coated with 1.2 µg of the monoclonal P III P 296, while adding 0 ,1 ml of phosphate

buffer saline solution (phosphate buffered saline, PBS) at room temperature for 2 hours. The test tubes are then washed twice with 1 ml of PBS each time and decanted. Then 200 µl of 125-J-labeled monoclonal P III P antibody (containing 40 ng of antibody) are placed in the tubes and they are then incubated for hours at room temperature. The radioactivity of the antibody-antigen-<12>^J-antibody complexes bound to the test tube wall is determined after washing twice with 1 ml of PBS each time and subsequent decantation in a scintillation measuring apparatus.

By comparison with an adjustment curve for the setting of which standards with different amounts of procollagen peptide (type III) are used, the concentration of procollagen peptide (type III) in the unknown sample solution can then be determined. Figure 2 shows the adjustment curve of the radioimmunometric analysis (B/T means: antibody bound/collected).

Example 8

Determination of molecular weight distribution of antigens reacting with PIIIP 296 in human serum. 1 ml of serum is divided per gel filtration chromatography over an allyl dextran cross-linked with N-N'-methylenebisacrylamide ["Sephacryl" S 300 column (1.6 x 130 cm)], equilibrated in "Phosphate buffered Saline" (PBS) with 0.04$ of a non- ionic detergent, for example a polyethoxylated sorbitol monolaurate (Tween 20), in fractions of 3.3 ml. Each 0.2 ml of the fractions is used in the radioimmunoassay according to example 4. Figure 3 shows the elution profile of the antigen determined by means of PIIIP 296 compared to the profile by means of polyclonal antibody determined reagent: Peak 4/4a corresponds to pN collagen and intact amino terminal

procollagen (type III)

Peak 5/5a corresponds to amino-terminal procollagen peptide

(type III)=(P III P)

Top 6/6a corresponds to Col 1 and breakdown products of the amino-terminal procollagen peptide (type III) with the same molecular weight as Col 1.

The concentration of the substances in the corresponding fractions can be determined using a procollagen peptide (type III) adjustment curve.

Claims (4)

1. Monoclonal antibody of the class IgG, characterized by the ability to react specifically with intact procollagen (type III), pN-collagen, ingested amino-terminal procollagen peptide (type III), however not binding to Col 1 and the breakdown products of the amino-terminal procollagen peptide (type III) with same molecular weight as Col 1. 2. Monoclonal antibody, characterized in that it is the antibody PIIIP 296 produced by the hybridoma cell line ECACC 87042308. 3. Hybridoma cell line that produces an antibody according to claims 1 and 2, characterized in that the cell line is the hybridoma ECACC 87042308 and in that it is formed by fusion of cells from a myeloma cell line and of lymphocytes from an animal previously immunized with procollagen peptide (type III). 4. Diagnostic preparation for determining the amount of procollagen peptide (type III) in body fluids, characterized in that it contains the monoclonal antibody according to claims 1 and 2 together with a diagnostically acceptable carrier.