2.8. ELISA Based Cytokine Quantification in Cell Supernatants

Cytokine levels (IL-1β, IL-6, IL-8, TNF-α, CCL2, CCL-5, CXCL10, and CXCL9) in supernatants of control and transfected cells were analyzed using human viral induced cytokines Multi-Analyte ELISArray kit (SABiosciences, Frederick, MD). Supernatants (1:2 diluted) were incubated in 96-well plates precoated with individual cytokine capture antibodies for 2 hours. After they were washed, biotin-conjugated cytokine detection antibodies were added for 30 minutes. Bound antibodies were detected with avidin-HRP and development solution. Color reactions were quantified at OD 450. Cytokine concentrations were determined by extrapolation from cytokine standard curves, according to the manufacturer's protocol. The experiments were repeated three times using supernatant from cells transfected with individual PB1-F2 constructs.