Characterization of an anti-decorin monoclonal antibody, and its utility.
6B6 is a monoclonal antibody raised against a purified small dermatan sulfate proteoglycan from human ovarian fibroma capsule, has Although it been widely used as an anti-decorin monoclonal antibody, its epitope has not yet been characterized at the molecular level. Here, we show that 6B6 is specific to decorin. The antibody recognized human, mouse, and bovine decorin core protein, but not biglycan. Using recombinant decorin domains, we determined that the epitope lies within the region of amino acid residues 50-65, termed the cysteine cluster region. Cross-reactivity among species further narrowed it down to a primary sequence of residues 57-65. We also established the conditions for immunostaining. 6B6 stained both frozen and fixed sections. Whereas the glycosaminoglycan chain of decorin inhibited access of the antibody in immunoblotting, pretreatment of tissue sections with chondrotinase ABC did not affect the intensity of staining, suggesting that the glycosaminoglycan chain is integrated and the Cys cluster region oriented outside of the collagen fibrils in the tissue. When 6B6 was applied to enzyme-linked immunosorbent assay, a concentration as low as 0.5 microg/ml of decorin was detectable by either direct or sandwich ELISA. 6B6 is thus a sensitive and reliable antibody to study functions of decorin from various aspects.
Sawada
Hiroo
H
Institute for Molecular Science of Medicine, Aichi Medical University Yazako, Nagakute, Aichi 480-1195, Japan.
Shinomura
Tamayuki
T
Kimata
Koji
K
Takeuchi
Jun
J
Tsuji
Takuo
T
Watanabe
Hideto
H
D000818:Animals; D000911:Antibodies, Monoclonal / Q000378:metabolism*; D058578:Biglycan; D002417:Cattle; D002478:Cells, Cultured; D058575:Decorin; D000939:Epitopes; D016326:Extracellular Matrix Proteins; D005347:Fibroblasts / Q000166:cytology / Q000378:metabolism; D006801:Humans; D051379:Mice; D010455:Peptides / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D011509:Proteoglycans / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D012867:Skin / Q000166:cytology / Q000378:metabolism / Q000473:pathology
D000911:Antibodies, Monoclonal; C546884:BGN protein, human; C546885:Bgn protein, mouse; D058578:Biglycan; C546878:DCN protein, human; C546879:Dcn protein, mouse; D058575:Decorin; D000939:Epitopes; D016326:Extracellular Matrix Proteins; D010455:Peptides; D011509:Proteoglycans; D011993:Recombinant Fusion Proteins