title: Trehalose conserves expression of bullous pemphigoid antigen 180 during desiccation and freezing.
abstract: Bullous pemphigoid antigen 180 (BP180) is targeted by autoantibodies in a variety of subepidermal blistering skin diseases. We have recently developed a simple, highly specific and sensitive immunofluorescence (IF) assay for the detection of circulating antibodies against BP180. This novel assay involves the expression of full-length (FL) BP180 in Sf21 insect cells that are then examined under IF microscopy after staining with anti-BP180 antibodies. Application of this assay as a routine diagnostic tool requires long-term storage of FL-BP180, which can result in substantial loss of expression. Here, we show that the disaccharide trehalose, a natural cryo- and lyoprotectant, is capable of preserving the FL-BP180 antigen expressed in Sf21 insect cells under various (dry) storage conditions including 40 degrees C, room temperature (RT), 4-8, -20, and -80 degrees C. The protective effect was dose-dependent reaching a maximum at about 200 mM trehalose. Trehalose was superior to other sugars or conventional cryoprotective agents (e.g. sucrose, myo-inositol, DMSO) in preventing greatly reduced antigen expression. Trehalose conserved the expression of both extra- and intracellular epitopes of FL-BP180. Interestingly, protection of the intracellular domain was only observed when trehalose was introduced into the cytosol. Trehalose significantly prolonged the storage time of FL-BP180 expressed in Sf21 insect cells, thus permitting the routine use of the IF assay in clinics for the detection of serum antibodies. The method described here has potential applications for the preservation of other transmembrane proteins.
authors.affiliation: Department of Biotechnology, Biocenter, University of Würzburg, Am Hubland, 97074, Würzburg, Germany.
authors.forename: Enno
authors.initials: E
authors.lastname: Schmidt
authors.forename: Arno
authors.initials: A
authors.lastname: Kromminga
authors.forename: Markus
authors.initials: M
authors.lastname: Kürschner
authors.forename: Heiko
authors.initials: H
authors.lastname: Zimmermann
authors.forename: Alisa D
authors.initials: AD
authors.lastname: Katsen
authors.forename: Eva Bettina
authors.initials: EB
authors.lastname: Bröcker
authors.forename: Detlef
authors.initials: D
authors.lastname: Zillikens
authors.forename: Ulrich
authors.initials: U
authors.lastname: Zimmermann
authors.forename: Vladimir L
authors.initials: VL
authors.lastname: Sukhorukov
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal; D001323:Autoantibodies / Q000097:blood; D001324:Autoantigens / Q000737:chemistry / Q000235:genetics*; D002352:Carrier Proteins; D002460:Cell Line; D015925:Cryopreservation; D003451:Cryoprotective Agents / Q000494:pharmacology*; D003598:Cytoskeletal Proteins; D003890:Desiccation; D000071821:Dystonin; D005615:Freezing; D015870:Gene Expression / Q000187:drug effects; D006801:Humans; D051379:Mice; D008856:Microscopy, Fluorescence; D009419:Nerve Tissue Proteins; D024041:Non-Fibrillar Collagens; D010391:Pemphigoid, Bullous / Q000235:genetics / Q000276:immunology; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics; D018411:Spodoptera; D014199:Trehalose / Q000494:pharmacology*; D000097805:Collagen Type XVII
chemical_list: D000911:Antibodies, Monoclonal; D001323:Autoantibodies; D001324:Autoantigens; D002352:Carrier Proteins; D003451:Cryoprotective Agents; D003598:Cytoskeletal Proteins; D000071821:Dystonin; D009419:Nerve Tissue Proteins; D024041:Non-Fibrillar Collagens; D011994:Recombinant Proteins; D014199:Trehalose; D000097805:Collagen Type XVII; C486268:DST protein, human