title: A simple fluorescence-based assay for quantification of the Toll-Like Receptor agonist E6020 in vaccine formulations.
abstract: Despite the generally accepted immunostimulatory effect of Toll-Like Receptor 4 (TLR4) agonists and their value as vaccine adjuvants, there remains a demand for fast and easy quantification assays for these TLR4 agonists in order to accelerate and improve vaccine formulation studies. A new medium-throughput method was developed for the quantification of the TLR4 agonist, E6020, independent of the formulation composition. The assay uses a fluorescent hydrazide (DCCH) to label the synthetic lipopolysaccharide (LPS) analog E6020 through its diketone groups. This novel, low-cost, and fluorescence based assay may obviate the need for traditional approaches that primarily rely on Fourier transform infrared spectroscopy (FTIR) or mass spectrometry. The experiments were performed in a wide diversity of vaccine formulations containing E6020 to assess method robustness and accuracy. The assay was also expanded to evaluate the loading efficiency of E6020 in poly(lactic-co-glycolic acid) (PLGA) micro-particles.
authors.affiliation: Departments of Pediatrics and Molecular Virology and Microbiology, National School of Tropical Medicine, Baylor College of Medicine, Houston, TX 77030, USA; Sabin Vaccine Institute and Texas Children's Hospital Center for Vaccine Development, Houston, TX 77030, USA.
authors.forename: Jeroen
authors.initials: J
authors.lastname: Pollet
authors.affiliation: Departments of Pediatrics and Molecular Virology and Microbiology, National School of Tropical Medicine, Baylor College of Medicine, Houston, TX 77030, USA; Sabin Vaccine Institute and Texas Children's Hospital Center for Vaccine Development, Houston, TX 77030, USA.
authors.forename: Leroy
authors.initials: L
authors.lastname: Versteeg
authors.affiliation: Departments of Pediatrics and Molecular Virology and Microbiology, National School of Tropical Medicine, Baylor College of Medicine, Houston, TX 77030, USA; Sabin Vaccine Institute and Texas Children's Hospital Center for Vaccine Development, Houston, TX 77030, USA.
authors.forename: Wanderson
authors.initials: W
authors.lastname: Rezende
authors.affiliation: Departments of Pediatrics and Molecular Virology and Microbiology, National School of Tropical Medicine, Baylor College of Medicine, Houston, TX 77030, USA; Sabin Vaccine Institute and Texas Children's Hospital Center for Vaccine Development, Houston, TX 77030, USA.
authors.forename: Ulrich
authors.initials: U
authors.lastname: Strych
authors.affiliation: Eisai Inc., RTP, Global Formulation Research, USA.
authors.forename: Fabian
authors.initials: F
authors.lastname: Gusovsky
authors.affiliation: Departments of Pediatrics and Molecular Virology and Microbiology, National School of Tropical Medicine, Baylor College of Medicine, Houston, TX 77030, USA; Sabin Vaccine Institute and Texas Children's Hospital Center for Vaccine Development, Houston, TX 77030, USA; Department of Biology, Baylor University, Waco, TX, USA.
authors.forename: Peter J
authors.initials: PJ
authors.lastname: Hotez
authors.affiliation: Departments of Pediatrics and Molecular Virology and Microbiology, National School of Tropical Medicine, Baylor College of Medicine, Houston, TX 77030, USA; Sabin Vaccine Institute and Texas Children's Hospital Center for Vaccine Development, Houston, TX 77030, USA; Department of Biology, Baylor University, Waco, TX, USA. Electronic address: bottazzi@bcm.edu.
authors.forename: Maria Elena
authors.initials: ME
authors.lastname: Bottazzi
mesh_terms: D000276:Adjuvants, Immunologic / Q000032:analysis*; D002623:Chemistry Techniques, Analytical; D005453:Fluorescence; D005456:Fluorescent Dyes / Q000378:metabolism; D005470:Fluorometry / Q000379:methods*; D006801:Humans; D010743:Phospholipids / Q000032:analysis*; D013194:Staining and Labeling / Q000379:methods; D051193:Toll-Like Receptors / Q000819:agonists; D014612:Vaccines / Q000737:chemistry*
chemical_list: D000276:Adjuvants, Immunologic; C000617976:ER-804053; D005456:Fluorescent Dyes; D010743:Phospholipids; D051193:Toll-Like Receptors; D014612:Vaccines
keywords: Adjuvant quantification; Fluorescent hydrazide labeling; TLR4 agonist