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A Direct Competitive Homogeneous Immunoassay for Progesterone – the Redox Quenching Immunoassay
Author(s) -
Ettlinger Julia,
Schenk Jörg A.,
Micheel Burkhard,
EhrentreichFörster Eva,
GajovicEichelmann Nenad
Publication year - 2012
Publication title -
electroanalysis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.574
H-Index - 128
eISSN - 1521-4109
pISSN - 1040-0397
DOI - 10.1002/elan.201200107
Subject(s) - immunoassay , chemistry , hapten , amperometry , detection limit , chromatography , quenching (fluorescence) , conjugate , monoclonal antibody , bifunctional , antibody , electrochemistry , biochemistry , fluorescence , electrode , catalysis , immunology , medicine , mathematical analysis , physics , mathematics , quantum mechanics
A direct competitive amperometric immunoassay format for the detection of haptens and proteins was developed. The method is based on the quenching of electroactivity of ferrocenium, which is coupled to the antigen and used as the primary reporter, upon binding to a monoclonal anti‐ferrocenium antibody, which is coupled to the detection antibody and used as a secondary reporter. A separation‐free progesterone immunoassay with a lower detection limit of 1 ng mL −1 (3.18 nmol L −1 ) in 1 : 2 diluted blood serum was realised by combining two bifunctional conjugates, a ferrocenium‐PEG‐progesterone tracer and a bioconjugate of one anti‐progesterone and one anti‐ferrocenium antibody. The immune complex is formed within 30 s upon addition of progesterone, resulting in a total analysis time of 1.5 min.