A monoclonal antibody that recognizes hyaluronic acid binding region of aorta proteoglycans.
A chondroitin sulfate-dermatan sulfate proteoglycan was isolated from bovine aorta intima by extraction of the tissue with 4 M guanidine hydrochloride. The proteoglycan was purified by CsCl isopycnic centrifugation followed by gel filtration and ion exchange chromatography. A monoclonal antibody C8F4 was developed to this core protein. The characteristics and specificity of the antibody were studied by an enzyme-linked immunosorbent assay (ELISA) using an alkaline phosphatase conjugated antibody (goat anti-mouse IgG). The antibody binding to the core protein was found specific and optimal at pH 7.0. The antibody recognizes either intact chondroitin sulfate-dermatan sulfate proteoglycan monomer, chondroitinase ABC digested monomer or chemically deglycosylated proteoglycan. Free chondroitin sulfates, keratan sulfate and hyaluronic acid did not compete for the antigenic sites in ELISA. Limited hydrolysis of the core protein by trypsin resulted in three peptides and only the peptide with a molecular weight M(r) = 40,000 was found capable of binding to hyaluronic acid. The antibody C8F4 recognized this hyaluronic acid binding peptide but did not recognize the other two peptides suggesting that the epitope(s) for this antibody is in the hyaluronic acid-binding region of the core protein. The antibody recognized the core proteins from bovine nasal cartilage proteoglycan and human aorta proteoglycan but did not recognize bovine aorta link protein, bovine serum albumin, human serum albumin, human transferrin, collagen Type I and fibronectin. The antibody was found useful to localize proteoglycans in atherosclerotic lesions in human aorta by immunohistochemical techniques.
Radhakrishnamurthy
B
B
Department of Medicine, Louisiana State University Medical Center, New Orleans.
Jeansonne
N
N
Tracy
R E
RE
Berenson
G S
GS
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D000918:Antibody Specificity; D001011:Aorta / Q000378:metabolism*; D001161:Arteriosclerosis / Q000378:metabolism; D001665:Binding Sites; D002417:Cattle; D002809:Chondroitin Sulfates / Q000276:immunology* / Q000378:metabolism*; D004797:Enzyme-Linked Immunosorbent Assay; D000939:Epitopes; D006801:Humans; D006820:Hyaluronic Acid / Q000276:immunology / Q000378:metabolism*; D051379:Mice; D008807:Mice, Inbred BALB C
D000911:Antibodies, Monoclonal; D000939:Epitopes; D006820:Hyaluronic Acid; D002809:Chondroitin Sulfates