title: Characterization of a novel recombinant hyaluronan binding protein for tissue hyaluronan detection.
abstract: Tumor necrosis factor-Stimulated Gene 6 protein (TSG-6) is a hyaluronan (HA)-binding glycoprotein containing an HA-binding Link module. Because of its well-defined structure, HA binding properties and small size, TSG-6 is an excellent candidate as an alternative to animal-derived HA-binding protein (HABP) for the detection of HA. The present work describes the generation and characterization of a novel recombinant HA-binding probe obtained by fusion of a modified TSG-6 Link module with mutationally inactivated heparin-binding sequence and the Fc portion of human IgG1 (TSG-6-ΔHep-Fc) for tissue HA detection in histological samples. Direct binding assays indicated strong binding of TSG-6-ΔHep-Fc to HA, with little residual binding to heparin. Histolocalization of HA in formalin-fixed, paraffin-embedded tissue sections using biotin-TSG-6-ΔHep-Fc resulted in hyaluronidase-sensitive staining patterns similar to those obtained with biotin-HABP, but with improved sensitivity. HA was detected in many human tissues, and was most abundant in soft connective tissues such as the skin dermis and the stroma of various glands. Digital image analysis revealed a linear correlation between biotin-HABP and biotin-TSG-6-ΔHep-Fc staining intensity in a subset of normal and malignant human tissues. These results demonstrate that TSG-6-ΔHep-Fc is a sensitive and specific probe for the detection of HA by histological methods.
authors.affiliation: Department of Research and Development, Halozyme Therapeutics, Inc., San Diego, California (LJ, LH, GW, QZ, ABG, GIF, PJ, HMS) ljadin@halozyme.com.
authors.forename: Laurence
authors.initials: L
authors.lastname: Jadin
authors.affiliation: Department of Research and Development, Halozyme Therapeutics, Inc., San Diego, California (LJ, LH, GW, QZ, ABG, GIF, PJ, HMS).
authors.forename: Lei
authors.initials: L
authors.lastname: Huang
authors.affiliation: Department of Research and Development, Halozyme Therapeutics, Inc., San Diego, California (LJ, LH, GW, QZ, ABG, GIF, PJ, HMS).
authors.forename: Ge
authors.initials: G
authors.lastname: Wei
authors.affiliation: Department of Research and Development, Halozyme Therapeutics, Inc., San Diego, California (LJ, LH, GW, QZ, ABG, GIF, PJ, HMS).
authors.forename: Qiping
authors.initials: Q
authors.lastname: Zhao
authors.affiliation: Department of Research and Development, Halozyme Therapeutics, Inc., San Diego, California (LJ, LH, GW, QZ, ABG, GIF, PJ, HMS).
authors.forename: Arnold B
authors.initials: AB
authors.lastname: Gelb
authors.affiliation: Department of Research and Development, Halozyme Therapeutics, Inc., San Diego, California (LJ, LH, GW, QZ, ABG, GIF, PJ, HMS).
authors.forename: Gregory I
authors.initials: GI
authors.lastname: Frost
authors.affiliation: Department of Research and Development, Halozyme Therapeutics, Inc., San Diego, California (LJ, LH, GW, QZ, ABG, GIF, PJ, HMS).
authors.forename: Ping
authors.initials: P
authors.lastname: Jiang
authors.affiliation: Department of Research and Development, Halozyme Therapeutics, Inc., San Diego, California (LJ, LH, GW, QZ, ABG, GIF, PJ, HMS).
authors.forename: H Michael
authors.initials: HM
authors.lastname: Shepard
mesh_terms: D000818:Animals; D019921:Biotinylation; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D005557:Formaldehyde / Q000494:pharmacology; D006493:Heparin / Q000378:metabolism; D006801:Humans; D018960:Hyaluronan Receptors / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D006820:Hyaluronic Acid / Q000378:metabolism*; D007141:Immunoglobulin Fc Fragments / Q000276:immunology; D009154:Mutation; D016612:Paraffin Embedding; D015202:Protein Engineering; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D016707:Tissue Fixation
chemical_list: D018960:Hyaluronan Receptors; D007141:Immunoglobulin Fc Fragments; D011994:Recombinant Proteins; D005557:Formaldehyde; D006820:Hyaluronic Acid; D006493:Heparin
keywords: Tumor necrosis factor-Stimulated Gene 6 protein; glycosaminoglycans; histological detection; hyaluronan; hyaluronan-binding protein; tissue localization