A modified ELISA accurately measures secretion of high molecular weight hyaluronan (HA) by Graves' disease orbital cells

Endocrinology. 2014 Feb;155(2):627-34. doi: 10.1210/en.2013-1890. Epub 2013 Dec 3.


Excess production of hyaluronan (hyaluronic acid [HA]) in the retro-orbital space is a major component of Graves' ophthalmopathy, and regulation of HA production by orbital cells is a major research area. In most previous studies, HA was measured by ELISAs that used HA-binding proteins for detection and rooster comb HA as standards. We show that the binding efficiency of HA-binding protein in the ELISA is a function of HA polymer size. Using gel electrophoresis, we show that HA secreted from orbital cells is primarily comprised of polymers more than 500 000. We modified a commercially available ELISA by using 1 million molecular weight HA as standard to accurately measure HA of this size. We demonstrated that IL-1β-stimulated HA secretion is at least 2-fold greater than previously reported, and activation of the TSH receptor by an activating antibody M22 from a patient with Graves' disease led to more than 3-fold increase in HA production in both fibroblasts/preadipocytes and adipocytes. These effects were not consistently detected with the commercial ELISA using rooster comb HA as standard and suggest that fibroblasts/preadipocytes may play a more prominent role in HA remodeling in Graves' ophthalmopathy than previously appreciated.

Publication types

  • Research Support, N.I.H., Intramural

MeSH terms

  • Adipocytes / drug effects
  • Adipocytes / metabolism*
  • Cell Differentiation / drug effects
  • Enzyme-Linked Immunosorbent Assay
  • Fibroblasts / drug effects
  • Fibroblasts / metabolism*
  • Graves Disease / metabolism*
  • Humans
  • Hyaluronic Acid / analysis*
  • Hyaluronic Acid / metabolism*
  • Interleukin-1beta / pharmacology
  • Receptors, Thyrotropin / metabolism


  • Interleukin-1beta
  • Receptors, Thyrotropin
  • Hyaluronic Acid