DK169583B1 - Monoclonal antibody, method for its preparation, hybridoma cell line producing the antibody, method for quantitative immunological determination of procollagen peptide (type III) with the antibody, and diagnostic composition containing the monoclonal antibody - 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

DK 169583 B1

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

Procollagen peptide (type III) is the amino terminal propeptide for collagen (type III), which is secreted extracellularly upon secretion of the procollagen (type III) molecule.

With a radioimmunological assay method, as described in European Patent Specification No. 4940, the concentration of this procollagen peptide in body fluids can be determined. Knowledge of the serum concentration of the peptide allows statements about the activity of fibrotic diseases, e.g. in the liver (H.

Rohde et al., Eur. J. Clin. Invest. 9, 451-459 (1979)).

However, accurate, selective determination of procollagen peptide (type III) in serum and other body fluids is not possible with the methods described previously, since the polyclonal antibodies used in these methods react with various serum antigens which in part are degradation products of procollagen peptide (type III), with different, low affinity (O. Niemelå et al., Clin. Chim. Acta 124. 39-44 (1982)). This means that the serum dilution curves and dilution curves for other 25 body fluids are not parallel to the standard curve set with pure procollagen peptide (type III) and therefore the antigen content of each unknown sample must be determined in multiple dilutions to measure antigen concentration above dilution 50. % s capture.

By the method of EP Patent Application No. 0.089,008, it is possible to solve this technical problem using antibodies having comparable activity for procollagen peptide (type III) and its degradation product Col 1. This method is determined intact 35 and disintegrated procollagen peptide (type III) together, which, however, leads to inaccuracy in the diagnostic statement as the normal and patient collective may strongly overlap.

Surprisingly, a monoclonal antibody has now been found that does not react with the degradation products of procollagen peptide (type III) present in body fluids.

With this monoclonal antibody, an immunological determination of the amino-terminal procollagen peptide (type III) has become possible, by which determination the peptide's degradation products are not determined, and are characterized by serum dilution curves, dilution curves for other body fluids and the standard curve.

The invention relates to: 1. A monoclonal antibody which has specific action against an epitope on intact procollagen (type III), pN collagen 15 and intact amino-terminal procollagen peptide (type III), but exhibits a weak reaction against Col 1 and degradation products of the amino terminal procollagen peptide (type III) having the same molecular weight as Col 1.

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

A method for producing a monoclonal antibody against amino-terminal procollagen peptide (III), wherein a) animals are immunized with amino-terminal procollagen peptide (type III), b) lymphocytes are recovered and fused with myeloma cells, c) hybrids are selected and cloned for (d) the antibody is recovered from said clones.

A method for quantitative immunological determination of procollagen peptide (type III) with antibodies, wherein a) a liquid sample containing amino-terminal procollagen peptide (type III) or procollagen (type III) is reacted with a monoclonal antibody of the invention, and b) the amount of the amino-terminal procollagen peptide (type III) or procollagen is determined via the formed antigen-antibody complex.

A diagnostic composition for quantitative immunological determination of procollagen peptide (type III) with antibodies, comprising a effective amount of the monoclonal antibody of the invention in admixture with a diagnostically acceptable carrier.

In the following, the invention is elucidated in detail, in particular the preferred embodiments, with reference to the drawing, in which fig. 1 shows the standard curves (1) and the serum dilution curves (2) with PIIIP 296 (a) and polyclonal antibodies (b), respectively; 2 shows the standard curve for a radioimmunometric assay; and FIG. 3 shows the reaction pattern for the monoclonal antibody PIIIP 296 and for polyclonal antibodies.

To prepare the monoclonal antibody, animals, preferably mammals, e.g. mice, rats, rabbits and guinea pigs, in the presence of adjuvant, are immunized with procollagen peptide (type III) isolated by the method of EP patent no. 4940. Mice, especially those of the SJL strain, are preferably used. With repeated secondary injections, e.g. at intervals of 4-8 weeks, the immune response is enhanced. A good result of the immunization is checked by determining the concentration of antibodies in the DK-169583 B1 4 radioimmunological binding assay (R. Timpl and L. Risteli, Immunochemistry of the extracullular matrix, H. Furthmayr, publisher, vol. 1, 199 (1982)). A few days before the fusion of the lymphocytes with a myeloma cell line, the animals are treated with procollagen 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 animal species mentioned above, but preferably from mice, especially with the cell line P3X63AG8.-653. Lymphocytes are advantageously fused with myeloma cell lines 10 from the same animal species. The fusion and further culturing of the cell clones is carried out in a manner known to those skilled in the art, the concentration of specific antibodies being determined in the above liquid in the cell culture by means of an immunological binding assay. From the cell clones arising from the fusion, suitable clones are selected for use in immunological methods. Particularly preferred is a cell line produced by fusion of lymphocytes from mice of the SJL strain against procollagen peptide (type III) with the mouse myeloma cell line P3X63AG8,653. This cell line is 20 deposited with the European Collection of Animal Cell Cultures (ECACC), PHLS Center for Applied Microbiology and Research, Porton Down, Salisbury, SP40JG, GB, under number 87042308.

The present monoclonal antibodies belong to the immunoglobulin group, preferably in the classes of 25 IgG, IgA and IgM proteins. Antibodies of the IgG class, especially of the IgG2b subclass, can be used with particular advantage.

The antibody in question is particularly surprising because its affinity for the antigen is higher than the affinity of polyclonal antibodies. Usually, one reaches the opposite result. The properties of the monoclonal antibody are made clear by the monoclonal antibody PIIIP 296 obtained from the cell line ECACC 87042308 when the antigens present in serum are broken down by their molecular weight via gel filtration chromatography and the fractions from the chromatography are used in radioimmunoassay. Thus, it appears that the antigenic fraction as determined by DK 169583 B1 analysis with polyclonal antibodies, which has the molecular weight of the degradation product Col 1, is not determined by the monoclonal antibody of the invention. In FIG. 3 of the drawing is shown the elution profile of human serum gel filtration chromatography 5, as the monoclonal antibody shows it, compared to the elution profile as polyclonal antibodies show it. Tip 4 / 4a corresponds to intact procollagen gene (type III), respectively. pN collagen (type III) (H. Rohde et al., Eur. J. Biochem. 135, 197 (1983)). Tip 5 / 5a corresponds to intact amino-terminal procollagen peptide (type III) (P III P), while tip 6 / 6a corresponds to Col 1 as well as to degradation products of the amino-terminal procollagen peptide (type III) of the same molecular weight as Col 1 (H. Rohde et al., Eur.

J. Biochem 135. 197 (1983)). The concentration of substances in the corresponding fractions can be determined by a standard curve for procollagen peptide (type III).

For the preparation of the antibodies in question, it is important that a suitable source of antigen extraction is available. As mentioned above, human 20 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 25 and / or immunoadsorption.

The present monoclonal antibodies may be used by various immunological methods, including all forms of radioimmunoassays, e.g. sequential saturation analysis or equilibrium analysis, optionally after labeling 30 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 by other competing and non-competing binding assays, such as fluorescence, enzyme, chemiluminescence or other immunoassays, including immunoradiometric assay (IRMA). The monoclonal antibody can therefore be used by immunological methods for iso- lation and characterization as well as for the quantitative determination of procollagen peptide (type III) in tissues and body fluids. Methods known to those skilled in the art are explored, in which a liquid sample containing procollagen peptide (type III) is reacted with the present monoclonal antibody, either in solution or on a solid support, and the amount of procollagen peptide (type III) is determined via the anti-gene antibody complex formed. It does not matter here whether or not procollagen peptide (type III) is still linked to the 10-amino-end portion of procollagen (type III). The disruptive degradation products of procollagen peptide (type III), in particular Col 1, heretofore, are not co-determined by the antibody in question.

In the following examples, the invention 15 is further illustrated. Percentages are by weight unless otherwise stated.

Example 1.

Preparation of procollagen peptide ftvpe ΙΙΙΪ (P III P>.

Procollagen peptide (type III) is prepared by the action of collagenase on procollagen (type III) at 37 ° C. Thereby, the peptide is not exposed to any denaturing agents.

To prepare larger amounts of the peptide, a modified method is used. All process steps until the action of the collagenase is carried out in cold stores. The various NaCl solution used for solubilization contains 0.05 M Tris-HCl, pH 7.4, 0.01 M EDTA, sodium azide (200 mg / ml) and the protease inhibitors phenylmethylsulfonyl fluoride (3 Mg / ml) and p-chloromercuribenzoate (3 Mg / ml).

30 3 kg of fetal calf skin are homogenized in 10 1 L M NaCl and extracted for 2 days. Dissolve collagen in common 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 xg, 20 minutes), washed twice with 2.5 M NaCl and dissolved again. , stirring overnight in 10 liters of 0.5 M NaCl. 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. Then the precipitate is suspended in 2 liters of 0.05 M Tris-HCl (pH 8.0) and heated after addition of 0.02 M 5 CaCl 2 for 20 minutes at 50 ° C and then incubated for 3 hours at 37 ° C together with 1500 units of bacterial collagenase (CLSPA, Worthington, USA) per grams of damp precipitate. After effecting the collagenase, the precipitate formed is separated by centrifugation and the solution is dialyzed against 0.005 10 M Tris-HCl, pH 8, 6.8 M urea and loaded onto a DEAE cellulose column (5.0 x 30 cm) which is equilibrated with the same buffer.

The proteins bound to the column are washed out with NaCl solutions, the concentration of which increases from 0 to 0.3 M.

15 The total elution amounts to 2 liters. The column flowing solution is tested for adsorption at 236 nm and its antigenic activity using antibodies specific for the amino-terminal segment of procollagen (type III). Usually, the last tip eluted for the column contains procollagen peptide (type III). The peptide is desalted by dialysis against distilled water and lyophilized. The further purification takes place on a column (2 x 120 cm) with agarose A 1.5 M, which is equilibrated with 1 M CaCl 2, 0.05 M Tris-HCl, pH 7.5.

Example 2.

Hvbridomaproduktion.

SJL strain mice are immunized intramuscularly with 5 µg procollagen peptide (type III) prepared according to Example 1, in the presence of Freund's complete adjuvant. At 30 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 Freund's incomplete adjuvant. Three days before the fusion, the immune response is enhanced by intraperitoneal injection of an additional 50 μg procol-35 layer 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. When culturing the fusion mixture in hypoxanthine aminopterin thymidine medium for a period of 2 weeks, spleen cell x P3X63AG8,653-5 hybrids are selected. To obtain a stable cell line, the resulting cell clones are subcloned several times. The resulting cell colonies are tested for antibody production by radioimmunological binding assay. The resulting cell line from which to recover the monoclonal antibody P III P 296 is deposited 10 with ECACC under number 87042308.

Example 3

Radioimmunobindinasafprøvning.

300 µl of the above cell culture liquid or another sample, e.g. ascites after culturing in the abdominal cavity of mice are incubated overnight with 100 μΙίΐβΓ of a solution of 125 I procollagen peptide (type III) (1 ng protein / 100 elites, prepared as described in EP 4940, Example 1). The antigen-antibody complexes formed are precipitated by the addition of sheep anti-mouse IgG serum or another animal species. After centrifugation and decanting of the above liquid, the amount of precipitated radioactivity is determined on the scintillation spectrometer.

Example 4.

Radioiimmunoafprovnina.

0.2 ml of the sample to be assayed or of procollagen peptide (type III) standard is incubated overnight at 48 ° C with a limiting amount of P III P 296 in the amount of labeled antigen 30 (in 0.1 ml buffer) and 0.1 ml of 125 I-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 6000). The precipitated antigen-antibody complexes are centrifuged (1500 x g) and, after frothing, radioactivity is determined in Tr scentillation spec- trometer.

By comparison with a standard curve for which the standard is used with different amounts of procollagen peptide (type III), the concentration of procollagen layer peptide (type III) in the unknown solution can be determined. Figure 1 of the drawing shows the standard curve (1) and serum dilution curves (2) with P III P 296 (a) compared to the method of European Patent Specification No. 4940 with polyclonal antibodies (b).

10

Example 5

Radioactive labeling of P III P 296.

0.2 ml of a solution of 0.2 mg of the P III P 296 monoclonal in 0.05 M phosphate buffer, pH 7.4, is filled into a polystyrene test tube (12 x 55 mm) and mixed with 100 MBq Na125I solution buffered with 10 eliters 0.5 M phosphate buffer, pH 7.4. After adding 50 µl of an aqueous solution of 20 µg chloramine T, mix for 1 minute. Then, the iodination reaction is terminated by the addition of 50 µl of an aqueous 20 solution of 20 µg sodium disulfite.

The unreacted Na125I is then separated from the 125i-labeled P III P 296 by chromatography on an anion exchanger.

The chromatographic fractions containing the purified 125 I-labeled antibodies are diluted with a solution of 20 25 g of "Tween® 20" and 14.6 g of Na2EDTA in a liter of 0.05 M Tris-HCl buffer, pH 8.0 , so that the concentration of the labeled antibody is 200 μg / liter.

Example 6

30 Antibody reward of test tube glass ♦

For fixation of the antibodies on polystyrene tubes (12 x 75 mm), 300 pliter of a solution of 4 mg monoclonal P III liter of 0.01 M sodium phosphate buffer, pH 6.4, and incubate overnight at room temperature. Then the antibody solution is aspirated. Then, in each glass, 500 μΙ of βΓ of a DK 169583 B1 10 solution is filled with bovine serum albumin in 0.05 M Tris-citrate buffer, pH 7.5, and incubated overnight at room temperature. Next, the solution is aspirated. The antibody coated tubes are dried over silica gel.

5

Example 7

Immunodiometric testing.

0.1 ml of the sample to be analyzed or 0.1 ml of procollagen (type III) standard is incubated at room temperature for 2 hours in polystyrene test tubes already coated with 1.2 Mg of monoclonal P III P 296, with the addition of 0.1 ml of phosphate buffered saline (PBS). Then the tubes are washed twice with 1 ml of PBS each time and decanted. Then the 200 Mliter 15 125 I-labeled monoclonal P III P antibody (containing 40 ng antibody) is loaded into the test tubes and incubated for hours at room temperature. The radioactivity of the antibody-antigen-125 I-antibody complexes bound to the test tube wall is determined in a scintillation meter after 2 washings each with 1 ml of 20 PBS and subsequent decantation.

By comparison with a standard curve for which standards using different amounts of procollagen peptide (type III) have been used, the concentration of procollagen peptide (type III) in the unknown sample solution 25 can be calculated. Figure 2 of the drawing shows the standard curve for the radioimmunometric assay. B / T means: antibody bound / total.

Example 8.

Determination of the molecular vein distribution of the human serum antigens with P III P 296.

1 ml of serum is partitioned by gel filtration chromatography over an allyl dextran cross-linked with N, N '-methylene bisacrylamide ("Sephacryl® S 300" column (1.6 x 130 cm)), equilibrated in 35 phosphate buffered saline (PBS) with 0.04% of a nonionic detergent, e.g. a polyethoxylated sorbitol monolayer (Tween ® 20), in 3.3 ml fractions. 0.2 ml of each of the fractions are used in the radioimmunoassay of Example 4. Figure 3 of the drawing shows the elution profile of the antigen determined by P III P 296 in Comparison 5 with the profile of the antigen determined by the polyclonal antibodies:

Tip 4 / 4a corresponds to pN collagen and intact, amino-terminal procollagen (type III), tip 5 / 5a corresponds to amino-terminal procollagen peptide (type 10 III) = (P III P), tip 6 / 6a corresponds to Col 1 and degradation products of the amino-terminal procollagen peptide (type III) having the same molecular weight as Col 1.

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

Claims (11)

1. Monoclonal antibody, characterized in that it has specific action against an epitope on intact procol layer (type III), pN collagen and intact amino-terminal procollagen peptide (type III), but exhibits a weak response to Col 1 and degradation products of the amino terminal procol layer peptide (type III) having the same molecular weight as Col 1. Monoclonal antibody according to claim 1, characterized in that it has specific action against an epitope 10 on the amino-terminal procollagen peptide (type III) and the pN-col layer, which epitope is not present on Col 1. The monoclonal antibody of claim 1 or 2, characterized in that it is assigned to the class IgG. Monoclonal antibody according to any one of claims 1 to 3, characterized in that it is formed by a hybridoma formed by fusion of cells from a myeloma line and by lymphocytes from a pre-collagen peptide (type III) immunized animal. 5. Monoclonal antibody P III P 296 from the hybridoma cell line ECACC 87042308. A hybridoma cell line producing an antibody according to claims 1-5, characterized in that it is formed by fusion of cells from a myeloma cell line and lymphocytes from a pre-collagen peptide (type III) immunized animal. 7. Hybridoma cell line ECACC 87042308. A method for producing a monoclonal antibody against amino-terminal procollagen peptide (type III), characterized in that 30 a) animals are immunized with amino-terminal procollagen peptide (type III), b) lymphocytes are recovered and fused with myeloma cells, c) hybrids are selected and an antibody having the properties set forth in any one of claims 1 to 5, and d) the antibody is recovered from said clones. DK 169583 B1 13 Method according to claim 8, characterized in that the hybridoma cell line ECACC 87042308 is used for carrying out step d). Method for quantitative immunological determination of procollagen peptide (type III) with antibodies, characterized in that a) a liquid sample containing amino-terminal procollagen peptide (type III) or procollagen (type III) is reacted with a monoclonal and b) the amount of the amino terminal procollagen peptide (type III) or procollagen is determined via the antigen-antibody complex formed. Diagnostic composition for use in the method 15 according to claim 10, characterized in that it consists of an effective amount of the monoclonal antibody according to one or more of claims 1-5 in admixture with a diagnostically acceptable carrier.