A simple fluorescence-based assay for quantification of the Toll-Like Receptor agonist E6020 in vaccine formulations

Vaccine. 2017 Mar 7;35(10):1410-1416. doi: 10.1016/j.vaccine.2017.01.077. Epub 2017 Feb 9.


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.

Keywords: Adjuvant quantification; Fluorescent hydrazide labeling; TLR4 agonist.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adjuvants, Immunologic / analysis*
  • Chemistry Techniques, Analytical*
  • Fluorescence
  • Fluorescent Dyes / metabolism
  • Fluorometry / methods*
  • Humans
  • Phospholipids / analysis*
  • Staining and Labeling / methods
  • Toll-Like Receptors / agonists
  • Vaccines / chemistry*


  • Adjuvants, Immunologic
  • ER-804053
  • Fluorescent Dyes
  • Phospholipids
  • Toll-Like Receptors
  • Vaccines