Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Patent
1991-09-27
1994-07-19
Scheiner, Toni R.
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
435 792, 435 794, 435 23, 436518, 436813, 5303911, 5303913, G01N 33573
Patent
active
053308948
DESCRIPTION:
BRIEF SUMMARY
TECHNICAL FIELD
The present invention relates novel monoclonal antibodies against a procollagenase whose corresponding collagenase cleaves Type I, Type II and Type III collagens, and a method for the assay of the procollagenase by enzymeimmunoassay utilizing thereof.
BACKGROUND ART
Collagenase, the enzyme which cleaves collagens, is widely distributed in organisms.
Since an elevated collagenase activity is noted in pathological tissues such as synovialis in rheumatoid arthritis, ulcerated cornea and osteoma tissues, it is beneficial to determine collagenase activity in pathological tissues and in body fluids for diagnosis of such disorders.
Collagenase is produced in a latent (inactive) form, procollagenase, and both of collagenase and procollagenase occur in tissues. Therefore, for the assay of collagenase activity, procollagenase has to be activated by a pretreatment with a protease such as trypsin or with a mercurial compound, etc. Moreover, as a large quantity of inhibitors of collagenase activity is present in tissues, a very complicated procedure is required in order to eliminate these inhibitors.
Several types of collagenases are known according to the types of their collagen substrates, and they are classified into the collagenase that cleaves type I, type II and type III collagens (interstitial collagens) (hereinafter referred to as interstitial collagenase), the collagenase that cleaves type IV collagen and type V collagen, and the like.
With regard to the monoclonal antibodies against the procollagenase of the interstitial collagenase (hereinafter referred to as "interstitial procollagenase"), 11 monoclonal antibodies have been disclosed, which were prepared by using as antigens a mixture of the interstitial procollagenases having a molecular weight of 52000 and 57000 [see Biochemistry, 27, 6751 (1988)]. However, the monoclonal antibodies of the present invention, which belong to the immunoglobulin in class and subclass G.sub.1, whose L-chain isotype is kappa and which have a collagenase inhibiting activity, have never been disclosed before.
DISCLOSURE OF INVENTION
BRIEF DESCRIPTION OF THE DRAWING
FIG. 1 illustrates an analytical curve from the assay of procollagenase utilizing antibody FERM BP-2701 (K5E1) as the immobilized monoclonal antibody and antibody FERM BP-2700 (K2F7) as the biotinylated anti-procollagenase monoclonal antibody.
Disclosure of Invention
The inventors have prepared novel monoclonal antibodies against a interstitial procollagenase having a molecular weight of 52000, and established a simple method for the assay of procollagenase by enzymeimmunoassay utilizing said antibodies, and then determined the concentration of procollagenase in human serum by the method. As the results, the inventors have found that the serum procollagenase concentration is significantly higher in patients of cancer rheumatoid arthritis (hereinafter abbreviated to "RA") and osteoarthritis (hereinafter abbreviated to "OA") than that in normal subjects, and thus accomplished the present invention.
It is an object of the present invention to provide novel monoclonal antibodies against a interstitial procollagenase. Another object of the present invention is to provide a simple method for the assay of the procollagenase by enzymeimmunoassay utilizing the novel monoclonal antibodies.
Determination of the procollagenase concentration in human serum using the assay method of the present invention is of great advantage in diagnosis of patients' disorders with elevated collagenase activity, such as cancer, RA, OA and the like.
Described below are the method for preparation of the monoclonal antibodies of the present invention and the method for the assay of a procollagenase by enzymeimmunoassay using said antibodies.
Described first is the method for preparation of the monoclonal antibodies of the present invention.
The monoclonal antibodies of the present invention may be obtained through the following process, (1) to (6) . molecular weight of 52000
The interstitial procollagenase having the mole
REFERENCES:
patent: 4376110 (1983-03-01), David et al.
Bergmann et al., J. Clin. Chem. Clin. Biochem., vol. 27, pp. 351-359. (1989).
Birkedal-Hansen et al., "Monoclonal Antibodies to Human Fibroblast, etc." Biochemistry 1988, 27, 6751-6758.
Birkedal-Hasen et al., "Use of Inhibitory (Anti-Catalytic) Antibodies, etc.", Immunological Investivations, 18 (1-4), 211-224 (1989).
Crespo et al., "Monoclonal Antibodies Against Synovial Collagenase, etc." Collagen Rev. Res. vol. 1/1988, pp. 1-10.
Petersen et al., "Production of Procollagenase by Cultured Human Keratinocytes", The Journal of Bio. Chem., vol. 262, No. 2, pp. 835-840 (1987).
Inoue Shintaro
Iwamoto Seiichi
Ohmoto Hiroshi
Kanebo Ltd.
Scheiner Toni R.
Wolski Susan C.
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