The monoclonal antibody 3/26 recognizes mouse complement protein activated C3 fragments C3b, iC3b, and C3c. The complement system is an important factor in innate immunity. The third complement component, C3, is central to the classical, alternative and lectin pathways of complement activation. C3 is the most abundant protein of the complement system with serum protein levels of about 1.3 mg/ml. Proteolysis by certain enzymes results in the cleavage of C3 into C3a and C3b. C3b becomes attached to immune complexes and is further cleaved into iC3b, C3c, C3dg and C3f. Activation products of the complement cascade contain neo-epitopes that are not present in the individual native components.
The synthesis of C3 is tissue-specific and is modulated in response to a variety of stimulatory agents. An inherited deficiency of C3 predisposes the person to frequent assaults of bacterial infections. In ulcerative colitis, and idiopathic chronic inflammatory bowel disease, the deposition of C3 in the diseased mucosa has been reported.
The monoclonal antibody 3/26 preferably recognizes cleaved C3 fragments C3b, iC3b, and C3c. In case of an acute inflammatory reaction lots of C3 are processed into the products recognized by 3/26 and is as such useful as marker for inflammatory reaction. In chronic inflammatory conditions minimal reactivity with 3/26 may observed. In such cases primarily the C3dg product resides at the place of inflammation (C3c being cleared) which is not recognized by antibody 3/26. The chronic processing/activation of C3 is taking place at a lower level, which would reduce detection of the C3 fragments C3b, iC3b, and C3c.
Flow cytometry, Frozen sections, Functional studies, Immuno assays, Immuno fluorescence, Western blot
FC: Antibody 3/26 stains deposited C3 fragments on the surface of cells. As positive control splenocytes were used and as negative control isotype control rat IgG2a. (Ref.1)
W: A non-reduced sample treatment and SDS-PAGE was used. The band size is ~185 kDa (Ref.1).
IHC-F: Tissue sections were fixed in acetone and pretreated with 3% hydrogen peroxide in methanol at RT to quench endogenous peroxidases. As positive control kidney derived from mice treated with anti-glomerular basement membrane antibodies was used and as negative control C3-/- mice kidney (Ref.1).
FS: The monoclonal antibody 3/26 inhibits the hemolytic activity of mouse complement in a dose-dependent manner. The biological activity of the antibody can be defined as the amount of antibody necessary for inhibition of hemolysis (Ref.1).
W: Blot is blocked with skimmed milk and incubated with culture supernatant (1:10) for 1h at RT
For immunohistochemistry, 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.
Zymosan activated mouse serum