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.
Bjerner
Johan
J
Central Laboratory, Norwegian Radium Hospital, Montebello, Oslo, Norway. johan.bjerner@klinmed.uio.no
Olsen
Kari Hauge
KH
Børmer
Ole P
OP
Nustad
Kjell
K
D000818:Animals; D000910:Antibodies, Heterophile / Q000097:blood / Q000276:immunology; D000911:Antibodies, Monoclonal / Q000276:immunology; D000918:Antibody Specificity / Q000276:immunology; D002272:Carcinoembryonic Antigen / Q000032:analysis* / Q000097:blood / Q000276:immunology*; D031541:Fluorescence Resonance Energy Transfer; D015200:Fluoroimmunoassay / Q000379:methods / Q000592:standards; D006801:Humans; D007136:Immunoglobulins / Q000276:immunology; D051379:Mice; D015203:Reproducibility of Results
D000910:Antibodies, Heterophile; D000911:Antibodies, Monoclonal; D002272:Carcinoembryonic Antigen; D007136:Immunoglobulins