Application of protein A-rosette assay for screening of monoclonal antibodies to human complement regulatory proteins.
Mice were immunized with purified membrane cofactor protein (MCP) and its monoclonal antibodies were screened by protein A(PA)-rosette assay. In this assay, the culture supernatants of hybridoma cells were layered over fixed MCP-bearing cells, and after washing, PA-coated sheep erythrocytes were applied as an indicator to these MCP-bearing cells. No purified antigen was therefore required throughout the screening. More than 300 of the supernatants harvested were successfully examined within 6 h. Each resultant antibody consisted of a single subclass of IgG, and reacted only with MCP in both transblotted and surface-labeled materials. The sensitivity of this assay was then assessed with these purified antibodies. As little as 0.5 micrograms of IgG1 or 0.01 micrograms of IgG2a was found to be detectable with more than 30% rosette formation. There were variations among cell lines in the sensitivity to the PA-rosette assay and the sensitivity did not correlate with the quantity of MCP surface expression in any of the cell lines. K562 gave the lowest background (nonspecific rosette formation) and the best specificity for anti-MCP of the 20 MCP-positive cell lines tested. Cell lines suitable for the detection of monoclonal anti-decay-accelerating factor and anti-C3b/C4b receptor were also examined and CCRF-SB and HSB2, and peripheral blood granulocytes, were found to be proper cell lines for screening the decay-accelerating factor and C3b/C4b receptor, respectively. Clones for anti C3b/C4b receptor were successfully obtained using granulocytes by the PA-rosette assay. This method needs no purified antigen and facilitates the rapid screening and purification of positive clones against cell-surface complement regulatory proteins.
Seya
T
T
Department of Immunology, Center for Adult Diseases, Osaka, Japan.
Hara
T
T
Uenaka
A
A
Nakayama
E
E
Akedo
H
H
D000911:Antibodies, Monoclonal / Q000032:analysis* / Q000302:isolation & purification; D015703:Antigens, CD; D001345:Autoradiography; D018958:CD55 Antigens; D002999:Clone Cells / Q000276:immunology; D003179:Complement C3b; D003165:Complement System Proteins; D004589:Electrophoresis, Disc; D004591:Electrophoresis, Polyacrylamide Gel; D006386:Hemagglutination Tests; D006801:Humans; D006825:Hybridomas / Q000276:immunology; D015151:Immunoblotting; D051916:Membrane Cofactor Protein; D008562:Membrane Glycoproteins / Q000276:immunology*; D008565:Membrane Proteins / Q000032:analysis; D011233:Precipitin Tests; D011951:Receptors, Complement / Q000032:analysis; D017463:Receptors, Complement 3b; D012397:Rosette Formation; D013205:Staphylococcal Protein A; D014407:Tumor Cells, Cultured
D000911:Antibodies, Monoclonal; D015703:Antigens, CD; C497857:CD46 protein, human; D018958:CD55 Antigens; D051916:Membrane Cofactor Protein; D008562:Membrane Glycoproteins; D008565:Membrane Proteins; D011951:Receptors, Complement; D017463:Receptors, Complement 3b; D013205:Staphylococcal Protein A; D003179:Complement C3b; D003165:Complement System Proteins