title: A modified ELISA accurately measures secretion of high molecular weight hyaluronan (HA) by Graves' disease orbital cells.
abstract: 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.
authors.affiliation: Laboratory of Endocrinology and Receptor Biology, National Institute of Diabetes and Digestive and Kidney Diseases, Bethesda, MD 20892.
authors.forename: Christine C
authors.initials: CC
authors.lastname: Krieger
authors.forename: Marvin C
authors.initials: MC
authors.lastname: Gershengorn
mesh_terms: D017667:Adipocytes / Q000187:drug effects / Q000378:metabolism*; D002454:Cell Differentiation / Q000187:drug effects; D004797:Enzyme-Linked Immunosorbent Assay; D005347:Fibroblasts / Q000187:drug effects / Q000378:metabolism*; D006111:Graves Disease / Q000378:metabolism*; D006801:Humans; D006820:Hyaluronic Acid / Q000032:analysis* / Q000378:metabolism*; D053583:Interleukin-1beta / Q000494:pharmacology; D011989:Receptors, Thyrotropin / Q000378:metabolism
chemical_list: D053583:Interleukin-1beta; D011989:Receptors, Thyrotropin; D006820:Hyaluronic Acid