The monoclonal antibody 2A1 recognizes rat C5b-9. The antibody was shown to compete with antibodies to human C9 for its binding site on the C5b-9 complex, indicating that the reactive epitope is located on the C9 molecule. C5b-9 membrane attack complexes are assembled from five precursor molecules in the serum. Proteolytic cleavage of C5 by C5 convertase generates C5b which initiates assembly of the C5b-9 complex. The last step of C5b-9 complex formation involves polymerization of C9 which accompanies insertion of the complex into the cell membrane. During formation of C5b-8 and C9 polymerization, neoantigens are generated which are unique to the C5b-9 complex and are not present on any of the individual native complex components. The complement regulatory proteins CD59 and complement S-protein can both prevent C5b-9 insertion into the cell membrane. The formed SC5b-9 complex is unable to attach to cells and is cytolytically inactive.C5b-9 is involved in the progression of chronic proteinuric renal disease by mediating continuous tubulointerstitial damage. Early tubulointerstitial injury in the remnant kidney can be improved when C5b-9 complex forming is abrogated.
The monoclonal antibody 2A1 was raised against a rat C5b-9 neoantigen. Monoclonal antibody 2A1 can be used as a coating antibody to detect C5b-9 in plasma and urine samples.
Flow cytometry, Frozen sections, Immuno assays, Immuno fluorescence, Paraffin sections
IA: plates were coated with 30μg/ml in 50mM carbonatebuffer, pH10.6 for 16h at 4°C.(Ref.1)
IHC-P: Tissue sections fixed in formalin were pretreated with protease type XXIV for 10 minutes at 37⁰C before incubation (Ref.6).
IHC-F: Tissue sections were fixed in acetone for 10 minutes at room temperature before incubation (Ref. 3).
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.
Glomeruli of rats treated with anti-Thy-1.1 antibodies