Circulating EDA, as determined by AlphaLisa, following pre-depletion with EctoD2, protein A, or EDAR-Fc. (a) Fetal calf serum (FCS), serum from an adult subject with XLHED, either pure (HED) or spiked with Fc-EDA (50 ng/mL; HED+), and serum from subject P6 were depleted twice with EctoD2 (anti-EDA)-coupled beads (+) or TACI-Fc-coupled beads (-). EDA in flow-through fractions was measured by AlphaLisa. (b) Same as (a) except that serum was depleted four times with Protein A-coupled beads (+) or Sepharose 6B beads (-). The beads were then acid-eluted and released immunoglobulins were quantified. No immunoglobulin was detected in the 4th elution of Protein A, indicating successful target depletion. There was no retention of immunoglobulins on Sepharose 6B. (c,d) Same as (a) except that sera were depleted once with EDAR-Fc (+) or with TACI-Fc (-) and that serum from an adult subject with XLHED was spiked with Fc-EDA at a concentration of 5 ng/mL (HED+). Measurements were conducted in duplicates. Adult, healthy adult control.