BACKGROUND: There are numerous potential sources of interference in immunoassays. Our aim was to identify the blood component that causes negative interference in cardiac troponin I (cTnI) immunoassays based on antibodies against the central part of cTnI. METHODS: We isolated an interfering factor (IF) from a sample with low recovery of added cTnI, using several consecutive purification steps: caprylic acid precipitation, ammonium sulfate precipitation, and purification on Cibacron Blue gel and protein G columns. Purified IF was identified by gel electrophoresis and mass spectrometric analysis of protein bands. For the direct detection of human antibodies to cardiac troponin in serum samples, we developed immunoassays using three different anti-human immunoglobulin antibodies and measured troponin antibodies in samples with low and normal cTnI recovery. RESULTS: Treatment with caprylic acid did not precipitate IF, but IF precipitated at 40% ammonium sulfate saturation. IF bound to a Cibacron Blue gel column, from which it was eluted with a linear salt gradient; it also bound to protein G. Gel electrophoresis of purified IF showed two major bands with molecular masses corresponding to the heavy (approximately 50 kDa) and light chains (approximately 25 kDa) of immunoglobulin, and their identities were confirmed by mass spectrometry. The presence of troponin-specific autoantibodies was confirmed in samples with low recoveries of cTnI by three different immunoassays. The median signals were significantly higher in 10 samples with low recovery than in 10 samples with normal recovery of cTnI (P < or = 0.007). CONCLUSIONS: Circulating autoantibodies to cTnI or other proteins of the troponin complex can be a source of negative interference in cTnI immunoassays.
BACKGROUND: There are numerous potential sources of interference in immunoassays. Our aim was to identify the blood component that causes negative interference in cardiac troponin I (cTnI) immunoassays based on antibodies against the central part of cTnI. METHODS: We isolated an interfering factor (IF) from a sample with low recovery of added cTnI, using several consecutive purification steps: caprylic acid precipitation, ammonium sulfate precipitation, and purification on Cibacron Blue gel and protein G columns. Purified IF was identified by gel electrophoresis and mass spectrometric analysis of protein bands. For the direct detection of human antibodies to cardiac troponin in serum samples, we developed immunoassays using three different anti-human immunoglobulin antibodies and measured troponin antibodies in samples with low and normal cTnI recovery. RESULTS: Treatment with caprylic acid did not precipitate IF, but IF precipitated at 40% ammonium sulfate saturation. IF bound to a Cibacron Blue gel column, from which it was eluted with a linear salt gradient; it also bound to protein G. Gel electrophoresis of purified IF showed two major bands with molecular masses corresponding to the heavy (approximately 50 kDa) and light chains (approximately 25 kDa) of immunoglobulin, and their identities were confirmed by mass spectrometry. The presence of troponin-specific autoantibodies was confirmed in samples with low recoveries of cTnI by three different immunoassays. The median signals were significantly higher in 10 samples with low recovery than in 10 samples with normal recovery of cTnI (P < or = 0.007). CONCLUSIONS: Circulating autoantibodies to cTnI or other proteins of the troponin complex can be a source of negative interference in cTnI immunoassays.
Authors: S Lachtermacher; B L B Esporcatte; F Montalvão; P C Costa; D C Rodrigues; L Belem; A Rabischoffisky; H C C Faria Neto; R Vasconcellos; S Iacobas; D A Iacobas; H F R Dohmann; D C Spray; R C S Goldenberg; A C Campos-de-Carvalho Journal: Braz J Med Biol Res Date: 2010-03-06 Impact factor: 2.590
Authors: Anna Bangert; Martin Andrassy; Anna-Maria Müller; Mariella Bockstahler; Andrea Fischer; Christian H Volz; Christoph Leib; Stefan Göser; Sevil Korkmaz-Icöz; Stefan Zittrich; Andreas Jungmann; Felix Lasitschka; Gabriele Pfitzer; Oliver J Müller; Hugo A Katus; Ziya Kaya Journal: Proc Natl Acad Sci U S A Date: 2015-12-29 Impact factor: 11.205
Authors: Eduardo M Vilela; Rita Bettencourt-Silva; J Torres da Costa; Ana Raquel Barbosa; Marisa P Silva; Madalena Teixeira; João Primo; Vasco Gama Ribeiro; José Pedro L Nunes Journal: Ann Transl Med Date: 2017-08