A Serum Bactericidal Assay for the Complement-Mediated Bactericidal Activity of Antibodies

Take a serially diluted serum sample containing monoclonal antibodies against lipopolysaccharides, or LPS — an outer membrane component of gram-negative bacteria.

Introduce gram-negative bacteria. Serum antibodies bind to bacterial LPS.

Add complement proteins. C1 complex binds to LPS-bound antibodies, forming an enzymatically active C1.

Activated C1 cleaves C4 and C2 to form C3 convertase, which cleaves C3 to form C5 convertase. The convertase cleaves C5 to produce C5b, which recruits C6 and C7. The resulting complex inserts into the outer membrane and associates with C8 and multiple C9, forming a pore.

Outer membrane damage destabilizes the inner membrane, causing cell lysis.

Spot the mixture onto an agar plate and spread. Incubate for bacterial growth. Overlay the plate with agar containing Triphenyl tetrazolium chloride, or TTC, which gets reduced by microbial dehydrogenases, imparting a red color to bacterial colonies.

Colony number increases with sample dilution, indicating reduced bactericidal activity with lower antibody concentration.