The monoclonal antibody JC4 is specific for Cytotoxic necrotizing factor type 1 and the highly related Cytotoxic necrotizing factor type 2 (CNF1 and CNF2) of uropathogenic Escherichia coli. CNF1 and 2 belong to a family of bacterial toxins that target the small GTP-binding Rho proteins that regulate the actin cytoskeleton. Members of this toxin family typically inactivate Rho; however, CNF1 and the CNF2 activate Rho by deamidation. CNF1 is more frequently associated with E.coli strains that cause extraintestitinal infections in humans, particularly those of the urinary tract (such as cystitis, pyelonephritis and prostatitis). In CNF1-producing uropathogenic E. coli strains, CNF1 is chromosomally encoded and typically resides on a pathogenicity island that also contains hemolysin and P fimbria- related genes. Both CNF1 and the highly related, plasmid-encoded CNF2 are monomeric, cytoplasmic toxins of approximately 115 kDa. CNF1 can be structurally organized into three functional domains the N-terminal binding domain, central and the C-terminal domain. The latter exhibits the catalytic activity of the toxin. Monoclonal antibody JC4 recognizes an epitope between amino acids 169 to 191 of the N-terminal binding domain. JC4 neutralizes only CNF1.
Functional studies, Immuno assays, Western blot
For 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 1:10. For inhibition of biological activity in vitro dilutions have to be made according to the amounts of CNF1 or CNF2 to be inactivated.