An In Vitro Method to Determine the Anti-Apoptotic Activity of Antibodies against TNFα

Take a multi-well plate containing mouse fibroblasts in serum-deprived media.  Add serial dilutions of TNFα-specific antibodies.

Add TNFα — an inflammatory cytokine.

Incubate. Serum deprivation induces cellular stress, making the cells susceptible to TNFα-mediated apoptosis.    

TNFα binding to its receptors on the serum-deprived cells triggers intracellular signaling cascades, activating the caspase cascade and inducing apoptosis.

However, antibody binding to TNFα prevents its receptor binding, inhibiting caspase activation.

Post-incubation, add a reagent mixture containing detergent, a proluminescent caspase substrate, and a luciferase enzyme along with ATP and magnesium ions. Mix and incubate.

The detergent permeabilizes the membranes, releasing caspases from TNFα-induced cells.

The bioluminescent substrate contains a caspase recognition site. Specific caspases cleave the substrate, generating aminoluciferin.

Luciferase oxidizes its substrate, aminoluciferin, resulting in light emission.

Measure luminescence intensity to detect a decline in luminescence with increasing antibody concentrations, indicating the potency of the TNFα specific-antibodies to neutralize TNFα and prevent TNFα-induced apoptosis.