Purified hAPRIL.01A and hAPRIL.03A bind APRIL with high affinity to APRIL and block binding to its receptors BCMA and TACI. (A) An ELISA in which soluble FLAG-hAPRIL was captured by rabbit anti-FLAG antibody and reactivity for purified Aprily-5 (A-5), hAPRIL.01A (hA.01A) and hAPRIL.03A (hA.03A) purified was measured. 50% effective concentration values were extrapolated from this ELISA. (B) Binding constants and equilibrium constants for both antibodies were analyzed using bio-light interferometry using the Octet system. (C) ELISA to examine the ability of hAPRIL.01A and hAPRIL.03A to block the binding of soluble FLAG-hAPRIL to adsorbed BCMA-Fc or TACI-Fc (both coated at 0.5 μg/mL). Aprily-5, a nonantagonistic anti-APRIL antibody, was used as a control. Antibodies were titrated by 5-fold dilution steps starting from 5 μg/mL.