Article Title: A Broad-Spectrum Infection Diagnostic that Detects Pathogen-Associated Molecular Patterns (PAMPs) in Whole Blood
Figure Lengend Snippet: Development of the FcMBL-based infection diagnostic. (a) Schematic representation of the FcMBL ELLecSA methodology in which PAMPs or whole pathogens contained in blood are captured using FcMBL-coated magnetic microbeads (FcMBL-beads) in combination with applied magnetic fields, and detection of bound material by application of recombinant human MBL linked to horse radish peroxidase (rhMBL-HRP), followed by addition of the chromogenic substrate, tetramethylbenzidine (TMB), which generates a positive signal in infected blood samples. (b) Mannan standard curve showing FcMBL binding to mannan measured as optical density at 450 nm, which was used to determine PAMP units (1 PAMP unit is equivalent to FcMBL binding to 1 ng/ ml mannan). Graph showing detection of the carbohydrate PAMP LPS (c) when diluted in whole blood using the FcMBL ELLecSA. Graphs showing enhanced detection of Klebsiella oxytoca (d), Klebsiella pneumonia (e), Enterobacter cloacae (f), and Escherichia coli (g) bacteria using the FcMBL ELLecSA in combination with (dark grey) or without (light grey) mechanical disruption (30 Hz for 10 min in a beadmill) or treatment with the antibiotic cefepime (100 μg/ml) (f) or amikacin (250 μg/ml) for 4 h (g) (* p
Article Snippet: The unbound rhMBL-HRP is removed by 4 washes with TBST 5 mM Ca2 + and PAMP detection is visualized by incubation in tetramethylbenzidine (1-stepTM Ultra TMB-ELISA, Cat no. 34028; Thermo Fisher, Waltham MA, USA).
Techniques: Infection, Diagnostic Assay, Recombinant, Binding Assay