Quantifying Protein Levels in Biological Samples

Protein concentrations in serum and in vaginal canal lysates were determined using enzyme-linked immunosorbent assay (ELISA). IgE concentration was measured using an IgE ELISA kit (Bethyl Laboratories, Montogomery, TX, USA). CXCL2 and IL-1β levels were quantified using cytokine specific ELISA kits (R&D Systems, Minneapolis, MN, USA) from whole cell lysates, as previously described [8 (link),9 (link)]. Absorbances for all ELISAs were measured at 450 nm and 570 nm using a PowerWave XZ microplate spectrophotometer (Biotek Instruments, Winooski, VT, USA). The recorded optical density measurements (OD) were then used to determine the protein concentration for each sample from a standard curve. Total protein concentrations in all samples were determined using a Detergent Compatible Assay (Bio-Rad, Hercules, CA, USA) following the manufacturers’ directions. Total protein concentrations were used to normalize concentrations of target proteins derived from ELISA assays.