Shuji Kodama

Monoclonal Antibodies to Bovine Collagenase Inhibitor

Hybridoma antibodies against bovine collagenase inhibitor were produced by fusion of myeloma cells NS-1 (P3-NS1-1) with spleen cells from mice hyperimmunized with collagenase inhibitor purified from the explant medium of bovine dental pulps. Hybridomas positive by an enzyme-linked immunosorbent assay (ELISA) for bovine collagenase inhibitor were cloned by the dilution method. Seventeen hybridomas producing antibodies were isolated, four of which also recognized purified human collagenase inhibitor in the ELISA. Using a monoclonal antibody-Sepharose affinity column, we easily purified both bovine and human collagenase inhibitors to homogeneity. They showed the same mobility on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, corresponding to a molecular mass of 32,000 daltons.

A Sandwich Enzyme Immunoassay for Collagenase Inhibitor Using Monoclonal Antibodies

A sandwich enzyme immunoassay for collagenase inhibitor was set up with a pair of monoclonal antibodies prepared against bovine dental pulp collagenase inhibitor. Two different combinations of the antibodies were found to be applicable to the immunoassay; one was for the determination of both bovine and human collagenase inhibitors, but the other was only for the bovine one. Minimum sensitivity was 1 pg/tube (3 pg/ml) for bovine collagenase inhibitor and 1.5 pg/tube (5 pg/ml) for human inhibitor. The levels of collagenase inhibitor in several normal human body fluids were measured by the immunoassay, and the values obtained were as follows: serum, 183 +/- 30 ng/ml (mean +/- SD); platelet-poor plasma, 68 +/- 13; cerebrospinal fluid, 60 +/- 13; amniotic fluid, 1,780 +/- 969; tear fluid, 418 +/- 145; mixed saliva, 316 +/- 166; urine, 7 +/- 8.

Immunohistochemical study on tissue inhibitors of metalloproteinases in normal and pathological human livers.

SummaryThe localization of tissue inhibitor of metalloproteinases (TIMP) in normal and pathological livers was examined by immunohistochemistry using monoclonal antibodies at the light microscopic level. In normal liver, immunoreactive TIMP was detected in smooth muscle cells and endothelial cells of blood vessels, fibroblasts, bile duct cells and Kupffer cells, indicating that TIMP is likely to be a general element of the liver. Immunoreactivity was observed in newly-formed blood vessels, proliferating bile ductules, and fibroblasts in the expanded portal area and fibrous septa of chronic active hepatitis and cirrhosis. TIMP was strongly stained in the capsule of hepatocellular carcinoma. The intensity of the immunoreaction in the capsule was generally greater than that in cirrhotic liver apart from the tumor mass. In three of five cases with hepatocellular carcinoma, endothelial walls in contact with tumor cells were positive.