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