Testing and validating a homogeneous immunometric assay for interference.

We analyzed 95 sera, demonstrating interference in a previous study, with the Kryptor homogeneous time-resolved fluorescence resonance energy transfer carcinoembryonic antigen (CEA) immunoassay (Brahms AG, Berlin, Germany). Only one serum differed, i.e., 6.0 microg/l for Kryptor vs. 13.3 microg/l for a microtiter plate in-house immunofluorometric assay (IFMA), using both aggregated mouse immunoglobulins as blocker and capture monoclonal antibody (Mab) F(ab')2-fragments to reduce interference. Sera were further analyzed with experimental IFMAs with intact capture Mabs and without blocker. The Kryptor-CEA assay Mab GFR44 (capture) had elevated results in 71% of sera with the Kryptor Mab G15 (tracer) or in 81% with our tracer Mab. G15 (capture) had 65% elevated results with GFR44 (tracer) or 99% with our tracer Mab. The latter was reduced from 99% to 62% by adding Kryptor blocker to the buffer or to 30% by adding our blocker. Finally, combining both assay Mabs and assay buffer from Kryptor still gave interference in 16% of sera. Kryptor-CEA assay results thus agreed with our in-house CEA assay results, showing no interference. As the Kryptor-CEA assay antibodies were sensitive to interference and the Kryptor-CEA assay buffer did not reduce interference as efficiently as our in-house assay buffer, the Kryptor-CEA assay format was crucial for the absence of interference.