Prominent immunogenicity of monosialosyl galactosylgloboside, carrying a stage-specific embryonic antigen-4 (SSEA-4) epitope in the ACHN human renal tubular cell line-a simple method for producing monoclonal antibodies against detergent-insoluble microdomains/raft.
The binding of Shiga toxin (Stx) to Gb3Cer in detergent-insoluble microdomains (DIM)/raft of the ACHN human renal tubular cell line causes the temporal activation of the Src-family kinase Yes [1]. As a strategy for examining signaling mechanisms in DIM/raft, monoclonal antibodies (MAbs) are reliable tools for characterizing the constituent molecules in these microdomains. Thus, we employed DIM/raft suspensions of ACHN cells as an immunogen to develop MAbs. Simply subcutaneous injections of ACHN DIM/raft could elevate the serum titer after several boosts. The first screening was performed using dot-blot immunostaining with culture supernatants on a polyvinylidene difluoride (PVDF) membrane, on which DIM/raft or their chloroform/methanol (C/M) (2:1, v/v) extracts were dot-blotted. The next screening was performed by flowcytometric analysis of ACHN cells treated with or without a permeabilizing reagent. Many of the clones (21/31 clones=68%) thus obtained were also found to recognize to lipid fractions of the DIM/raft. Strikingly, all of the 21 clones that reacted to the lipid fraction were found to recognize monosialosyl galactosylgloboside (MSGG) or GL7, which carries the SSEA-4 epitope. Using DIM/raft as immunogens may enable us to easily obtain MAbs for glycolipids.
Katagiri
Y U
YU
Department of Pathology, National Children's Medical Research Center, 3-35-31, Taisido, Setagaya-Ku, Tokyo 154-8509.
Ohmi
K
K
Katagiri
C
C
Sekino
T
T
Nakajima
H
H
Ebata
T
T
Kiyokawa
N
N
Fujimoto
J
J
D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis / Q000276:immunology*; D000941:Antigens / Q000737:chemistry / Q000276:immunology; D002460:Cell Line; D002499:Centrifugation, Density Gradient; D002855:Chromatography, Thin Layer; D000939:Epitopes / Q000737:chemistry / Q000276:immunology*; D005434:Flow Cytometry; D005915:Globosides / Q000737:chemistry / Q000276:immunology*; D006028:Glycosphingolipids / Q000737:chemistry / Q000276:immunology*; D006801:Humans; D006825:Hybridomas / Q000276:immunology; D015151:Immunoblotting; D007668:Kidney / Q000166:cytology / Q000276:immunology*; D021962:Membrane Microdomains / Q000737:chemistry / Q000276:immunology*; D051379:Mice; D012995:Solubility; D055746:Stage-Specific Embryonic Antigens; D014407:Tumor Cells, Cultured
D000911:Antibodies, Monoclonal; D000941:Antigens; D000939:Epitopes; D005915:Globosides; D006028:Glycosphingolipids; D055746:Stage-Specific Embryonic Antigens; C066584:stage-specific embryonic antigen-4