Anti-rhEPO antibody response: specificity and IgG subclasses. The baseline on each SPR-based biosensor immunoassay sensorgram represents a signal derived from EPO immobilized to the SPR chip surface, prior to injection of patient sample P1 at t = 0 seconds. “P1 binding” represents material remaining bound after injection of patient sample (as indicated). (A) Verification of rhEPO specificity of patient P1 serum. Injection of 1:10 diluted patient serum (upper line) compared with patient serum spiked with 10 μg/mL rhEPO (lower line), which resulted in 91% relative signal decrease compared to 2 minutes after end of serum injection, demonstrating rhEPO specificity of the P1 patient serum sample. (B) Antibody isotyping: injection of patient sample followed by serial injection of anti-IgM, anti-IgE, and anti-IgG. Only IgG shows significant signal increase (>300%, suggesting multiple binding of the polyclonal anti–human-IgG antibody), confirming ADA as an IgG class molecule. (C) Antibody subclass determination: injection of patient sample followed by serial injection of anti-IgG3: no signal increase (9% signal decrease 2 minutes after end of injection); anti-IgG2: minor increase (8% increase); and anti-IgG1 (strong signal increase of 61% confirming the presence of IgG1 in sample P1). (D) Antibody subclass determination: injection of patient sample followed by injection of anti-IgG4 (25% increase, confirming the presence of IgG4 in sample P1). inj, injection; RU, resonance units; s, seconds; t, time.