The monoclonal antibody 1H8 recognizes Complement Factor C3b/iC3b/C3d, it specifically binds human C3 as well as the breakdown products C3b, iC3b and C3dg. C3 plays a central role in the activation of complement system. Its activation is required for both classical and alternative complement activation pathways. People with C3 deficiency are susceptible to bacterial infection.
One form of C3-convertase, also known as C4b2a, is formed by a heterodimer of activated forms of C4 and C2. It catalyzes the proteolytic cleavage of C3 into C3a and C3b, generated during activation through the classical pathway as well as the lectin pathway. C3a is an anaphylotoxin and the precursor of some cytokines such as ASP, and C3b serves as an opsonizing agent. Factor I can cleave C3b into C3c and C3d, the latter of which plays a role in enhancing B cell responses. In the alternative complement pathway, C3 is cleaved by C3bBb, another form of C3-convertase composed of activated forms of C3 (C3b) and factor B (Bb). Once C3 is activated to C3b, it exposes a reactive thioester that allows the peptide to covalently attach to any surface that can provide a nucleophile such as a primary amine or a hydroxyl group. Activated C3 can then interact with factor B. Factor B is then activated by factor D, to form Bb. The resultant complex, C3bBb, is called the alternative pathway (AP) C3 convertase.
C3bBb is deactivated in steps. First, the proteolytic component of the convertase, Bb, is removed by complement regulatory proteins having decay-accelerating factor (DAF) activity. Next, C3b is broken down progressively to first iC3b, then C3c + C3dg, and then finally C3d. Factor I is the protease that performs these cuts with CR1 as cofactor. Clone 1H8 recognize separate, non-overlapping epitopes on C3 fragments. Levels of C3 in the blood may be measured to support or refute a particular medical diagnosis. For example, low C3 levels are associated with some types of kidney disease such as post-infectious glomerulonephritis and shunt nephritis.
Flow cytometry, Immuno precipitation, Western blot
FC: Antibody 1H8 stains the extracellular domain of C3b/iC3b/C3d. As positive control Balb/c thymus (activated with anti-Mouse T-Cells and Human Serum) was used and as negative control secondary antibody. (Ref.1)
W: A reduced sample treatment and SDS-Page was used. The band size is 195 kDa (Ref.2).
For flow cytometry and Western blotting, 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. For functional studies, in vitro dilutions have to be optimized in user’s experimental setting.