The monoclonal antibody 3D5 recognizes complement decay accelerating factor (DAF), also designated as CD55. Cells express on their surface several proteins which protect against complement attack, namely C receptor I (CR1), decay accelerating factor, membrane cofactor protein (MCP) and CD59. CR1, CD55 and MCP regulate the activation pathways of complement by either accelerating decay of the C3 and C5 convertase (CR1, CD55), or acting as cofactors for the serine protease factor I, which cleaves and irreversibly inactivates C3b (CR1, MCP).
Mouse CD55 is a 60 kDa transmembrane protein that binds C3b and C4b to inhibit formation and half-life of the C3 convertases. CD55 is broadly distributed among cells in contact with serum, including both haematopoietic and nonhaematopoietic cells. Although CD55 does not have an essential role in controlling hemolysis of erythrocytes, it has an important role in regulation of the deposition of C3 on nucleated cells.
Together with other complement regulators CD55 protects self-cells from autologous complement-mediated injury. CD55 cooperates with CD46 in circumventing autologous C3 deposition, while CD59 inhibits the pathway at the critical end-point.
Flow cytometry, Immuno assays, Paraffin sections, Western blot
IA: HM1115 was used as a detection antibody in a direct ELISA (plates coated with mouse CD55. The concentration of HM1115 used was 2μg/ml.
W: Proteins were separated by SDS-PAGE, transferred to nitrocellulose membranes and blocked with Tris-buffered saline containing 0.05% Tween 20 (TBST),3% non-fat dried milk and 3% BSA (Ref 1).
P: A mixture of 3D5 and another antibody was used. Colon segments were fixed overnight in 10% Formalin, embedded in paraffin blocks, and cut into 5 μm sections. Sections were
stained with H&E (Ref.5).
Dilutions to be used depend on detection system applied. It is recommended that users test the reagent and determine their own optimal dilutions. The typical starting working dilution is 1:50.