title: Total internal reflection (TIRF)-based quantification of procalcitonin for sepsis diagnosis--a point-of-care testing application.
abstract: A new, highly sensitive fluorescence immunoassay for a TIRF (total internal reflection)-based point-of-care testing (POCT) device was developed for the detection of procalcitonin (PCT), a specific and early marker for sepsis and microbial infections. The immunoassay was performed on a bench-top system that fulfilled all the necessary characteristics of a POCT application, including a short measurement time (<9 min), no sample pre-treatment requirements and application directly near patients. New rat monoclonal antibodies targeting PCT were screened and characterized. The best combinations of antibodies were then integrated into single-use cartridges, and the reduction of nonspecific binding was achieved by supplying suitable additives. Moreover, human recombinant PCT (hrPCT) for use as a standard was developed in the native form of hPCT in plasma (PCT1-116, PCT3-116). The assay achieves the required sensitivity range in human plasma to allow reliable differentiation between healthy persons and varying stages of infection severity (LOD=0.04 ng/mL; LOQ=0.12 ng/mL). Furthermore, the developed PCT assay can be applied in whole human blood with an adequate sensitivity (LOD=0.02 ng/mL; LOQ=0.09 ng/mL). To the best of our knowledge, this is the first diagnostic test for sepsis to use whole blood, which is a crucial requirement for POCT. We were able to detect native PCT in patient samples and showed a good correlation (R(2)=0.988) with the results of the Kryptor(®) device from BRAHMS, a state of the art device for the detection of PCT.
authors.affiliation: Research Unit Microbe-Plant Interactions, Helmholtz Zentrum München, German Research Center of Environmental Health (GmbH), Ingolstädter Landstraße 1, D-85764 Neuherberg, Germany.
authors.forename: Daniela
authors.initials: D
authors.lastname: Rascher
authors.affiliation: Institute of Structural Biology, Helmholtz Zentrum München, German Research Center of Environmental Health (GmbH), Ingolstädter Landstraße 1, D-85764 Neuherberg, Germany.
authors.forename: Arie
authors.initials: A
authors.lastname: Geerlof
authors.affiliation: Institute of Molecular Immunology, Helmholtz Zentrum München, German Research Center of Environmental Health (GmbH), Marchioninistraße 25, D-81377 Munich, Germany.
authors.forename: Elisabeth
authors.initials: E
authors.lastname: Kremmer
authors.affiliation: Research Unit Microbe-Plant Interactions, Helmholtz Zentrum München, German Research Center of Environmental Health (GmbH), Ingolstädter Landstraße 1, D-85764 Neuherberg, Germany.
authors.forename: Petra
authors.initials: P
authors.lastname: Krämer
authors.affiliation: Research Unit Microbe-Plant Interactions, Helmholtz Zentrum München, German Research Center of Environmental Health (GmbH), Ingolstädter Landstraße 1, D-85764 Neuherberg, Germany.
authors.forename: Schmid
authors.initials: S
authors.lastname: Michael
authors.affiliation: Research Unit Microbe-Plant Interactions, Helmholtz Zentrum München, German Research Center of Environmental Health (GmbH), Ingolstädter Landstraße 1, D-85764 Neuherberg, Germany.
authors.forename: Anton
authors.initials: A
authors.lastname: Hartmann
authors.affiliation: Research Unit Microbe-Plant Interactions, Helmholtz Zentrum München, German Research Center of Environmental Health (GmbH), Ingolstädter Landstraße 1, D-85764 Neuherberg, Germany. Electronic address: martin.rieger@helmholtz-muenchen.de.
authors.forename: Martin
authors.initials: M
authors.lastname: Rieger
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry; D015415:Biomarkers / Q000097:blood*; D015374:Biosensing Techniques / Q000295:instrumentation*; D002116:Calcitonin / Q000097:blood*; D015740:Calcitonin Gene-Related Peptide; D004867:Equipment Design; D006801:Humans; D007118:Immunoassay / Q000295:instrumentation; D019095:Point-of-Care Systems; D011498:Protein Precursors / Q000097:blood*; D051381:Rats; D012680:Sensitivity and Specificity; D018805:Sepsis / Q000097:blood / Q000175:diagnosis*
chemical_list: D000911:Antibodies, Monoclonal; D015415:Biomarkers; C000606589:CALCA protein, human; C000606591:Calca protein, rat; D011498:Protein Precursors; D002116:Calcitonin; D015740:Calcitonin Gene-Related Peptide
keywords: Evanescent field; FIA; POCT; Sepsis; Tirf; hrPCT