Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Patent
1989-06-23
1992-12-29
Kepplinger, Esther L.
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
435 792, 435 794, 435 723, 435 28, 436518, 436531, 436534, 436 64, 5303911, 5303913, G01N 3353, G01N 33574
Patent
active
051750848
DESCRIPTION:
BRIEF SUMMARY
FIELD OF THE INVENTION
The present invention relates to a method of the diagnosis for hepatic carcinoma by way of quantitation of collagenase inhibitor. More particularly the invention relates to a method for carrying out the diagnosis of hepatic carcinoma, by means of a sandwich enzyme immunoassay for human collagenase inhibitor using monoclonal antibodies to bovine collagenase inhibitor (tissue inhibitors of metalloproteinases: TIMP), on the basis of the fact that the amount of collagenase inhibitor in sera, plasmas or synovial fluids from patients with hepatic carcinoma is clearly higher than the amount of collagenase inhibitor in sera, plasmas or synovial fluids, respectively, from normal subjects. The enzyme immunoassay mentioned above is used to mean a method for determining collagenase inhibitor which is characterized in that there are used two different monoclonal antibodies which specifically bind to different antigenic determinants of collagenase inhibitor as an antibody to be bound to a solid phase support and an antibody to be labeled with an enzyme.
BACKGROUND ART
Collagenase inhibitor has been known to exist in human's and other animals' bones, skins, dental pulps, amniotic fluids, bloods and synovial fluids as well as in cultures of joint chondrocytes, synovial cells, fibroblasts derived from varied tissues and fibrosarcomatous cells. As means for determining the amount of collagenase inhibitor, methods have heretofore been known which are based on measurement of its biological activity. As described by Eisen et al. in J. Lab. Clin. Med. 75, 258-263 (1973) and also by Cawston et al. in Arthritis and Rheumatism 27, 285-290 (1984), however, it is impossible, with the heretofore known methods of determination, to assay sera, plasmas or synovial fluids for collagenase inhibitor activity since there exist in such fluids proteins, such as .alpha..sub.2 -macroglobulin, which interfere disturbingly with the measurement. Hayakawa, Iwata et al. have already developed a sandwich enzyme immunoassay (EIA) using monoclonal antibodies to bovine collagenase inhibitor, a method which enables specific quantitation of collagenase inhibitor with very small volumes of samples in a precise, straightforward and rapid manner (Japanese Patent Application No. Sho 62-42781). The present inventors have discovered that the level of collagenase inhibitor present in human sera, plasmas or synovial fluids apparently increases in association with affliction with hepatic carcinoma, and found that it is possible to make a diagnosis of hepatic carcinoma by determining, by the enzyme immunoassay mentioned above, the amount of collagenase inhibitor present in sera, plasmas or synovial fluids.
BRIEF DESCRIPTION OF DRAWINGS
FIG. 1 shows Western blotting patterns of bovine dental pulp collagenase inhibitor subjected to SDS-PAGE, obtained with different monoclonal antibodies;
FIG. 2 shows a standard curve for bovine dental pulp collagenase inhibitor, prepared with a solid phase 7-23G9 antibody-conjugate Fab'(7-6C1)-POD assay system;
FIG. 3 shows immunologically stained patterns obtained by subjecting human serum collagenase inhibitor to SDS-PAGE and then to Western blotting;
FIG. 4 shows a standard curve for human serum collagenase inhibitor, prepared with a solid phase 7-23G9 antibody-conjugate Fab'(7-6C1)-POD assay system;
FIG. 5 shows SDS-PAGE patterns of collagenase inhibitor purified from human sera;
FIG. 6 shows a standard curve prepared by sandwich assay for purified human serum collagenase inhibitor; and
FIG. 7 shows Western blotting patterns obtained when the eluted fraction obtained upon application of human sera onto an affinity column with IgG(7-23G9) antibody bound was subjected to SDS-PAGE followed by application of Fab'(7-6C1)-POD conjugate.
DISCLOSURE OF THE INVENTION
The present invention has been accomplished on the basis of the findings as mentioned above, and provides a method for diagnosing whether the subject is afflicted with hepatic carcinoma, which comprises carrying out an enzyme immunoassay by th
REFERENCES:
Kodama et al (1989) Columbus, Ohio 110 (19):445 "EIA of Collagenase Inhibitor in Blood and Other Samples".
Stricklin et al. (1986) Clinical Research 34(2):783A "Immunolocalization of collagenase inhibitor in normal skin and the stroma of basal cell carcinoma".
Eisen et al., J. Lab. Clin. Med. 75, 258-263 (1970).
Cawston et al., Arthritis and Rheumatism, 27, No. 3, 285-290 (1984).
Fundamentals of Comparative Embryology of the Vertebrates by Alfred F. Huettner, New York, The MacMillan Company, 1949, p. 222.
Voller et al, "Enzyme-Linked Immunosorbent Assay" in Manual of Clinical Laboratory Immunology, pp. 99-109 (1986).
Hayakawa Taro
Ichida Takafumi
Inoue Kyoichi
Iwata Kazushi
Kodama Shuji
Bidwell Carol E.
Fuji Yakuhin Kogyo Kabushiki Kaisha
Kepplinger Esther L.
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