Monoclonal antibody 2952 reacts with a neo-epitope on C5a (des-Arg) that is formed upon cleavage of C5 into C5a and C5b. C5 is involved in the activation of the lythic pathway within the complement system which is an important factor in innate immunity. The complement pathways can be divided in the activation pathways and lytic pathway. The activation pathways lead via C3 to the cleavage of the fifth complement component C5. C5a induces smooth muscle contraction, increases vascular permeability, causes degranulation of mast cells and basophils, and release of lysosomal enzymes. In addition C5a stimulates the directed migration of neutrophils, eosinophils, basophils and monocytes. Recent studies indicate the modulation of the acute-phase response in liver and an overall immune response by inducing synthesis of cytokines such as TNF-alpha IL-1beta, IL-6 and IL-8. General comment: Please notice that under given conditions it is known that C5 can expose epitopes normally only found in the cleaved activation products (ref.5).
Frozen sections, Functional studies, Immuno assays, Western blot
For immunohistology, 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.
IHC-P: No pretreatment was used on human tissue sections. Antibody was used at a concentration of 1 μg/ml. As positive control human granulocytes were used.
IHC-F: Tissue sections were fixed in Histochoice MB (Amresco) and blocked with DakoCytomation Protein Block. Pretreatment with levamisole solution was performed to quench endogenous phosphatases. As positive control drusen eye sections was used and as negative control sections lacking the first antibody (Ref.4).
FS: Antibody 2952 functions as a neutralizing antibody blocking the interaction between C5a and its receptor. The antibody was functionally tested by competitive binding studies using [125I]C5a and Bt2-cAMP differentiated U937 cells (Ref.2).The biological activity can be defined as the ability of antibody to displace C5a from U937 cell surface.
Positive control Human Granulocytes.
General comment: Please notice that under given conditions it is known that C5 can expose epitopes normally only found in the cleaved activation products (ref.5).