Isolation and characterization of two monoclonal antibodies that recognize remote epitopes on the cell-binding domain of human fibronectin.
Two monoclonal anti-fibronectin antibodies that inhibit fibronectin-mediated cell adhesion have been established and characterized. One antibody, FN12-8, inhibited attachment of rat kidney fibroblasts on the fibronectin-coated substrate in a concentration-dependent manner, attaining a maximal inhibition of greater than 85% at 850 micrograms/ml. Another antibody, FN30-8, caused about 70% inhibition at a concentration as low as 0.85 microgram/ml, although further increase of the antibody concentration did not significantly augment the inhibitory effect. Immunoblot analysis with defined proteolytic fragments revealed that both antibodies are directed to the cell-binding domain of fibronectin. The epitopes for these antibodies were further narrowed down using recombinant cell-binding fragments expressed in Escherichia coli. FN12-8 recognized the 11.5-kDa cell-binding fragment previously characterized by Pierschbacher et al. (1981, Cell 26, 259-267), suggesting that FN12-8 blocks the Arg-Gly-Asp (RGD) cell adhesion signal. FN30-8 could not bind this fragment but did recognize a longer cell-binding fragment containing additional greater than 111 amino acid residues upstream of the 11.5-kDa fragment. Since the RGD-dependent cell adhesion seems to require another signal located at a region 50-160 residues upstream of the 11.5-kDa fragment for full activity, FN30-8 may exert its inhibitory effect by blocking the latter signal.
Katayama
M
M
Pharmaceutial Research Laboratories, Takara Shuzo Company, Ltd., Shiga, Japan.
Hino
F
F
Odate
Y
Y
Goto
S
S
Kimizuka
F
F
Kato
I
I
Titani
K
K
Sekiguchi
K
K
D000596:Amino Acids / Q000032:analysis; D000911:Antibodies, Monoclonal / Q000032:analysis / Q000276:immunology / Q000302:isolation & purification*; D002448:Cell Adhesion / Q000187:drug effects; D000939:Epitopes / Q000032:analysis / Q000276:immunology*; D005353:Fibronectins / Q000032:analysis / Q000276:immunology* / Q000494:pharmacology; D006801:Humans
D000596:Amino Acids; D000911:Antibodies, Monoclonal; D000939:Epitopes; D005353:Fibronectins