Autodisplay of 60-kDa Ro/SS-A antigen and development of a surface display enzyme-linked immunosorbent assay for systemic lupus erythematosus patient sera screening Klaudia Petermann a , Stefan Vordenbäumen b , Jae-Chul Pyun c , Achim Braukmann a , Ellen Bleck b , Matthias Schneider b , Joachim Jose a,⇑ a Bioanalytics, Institute of Pharmaceutical and Medicinal Chemistry, Heinrich Heine University Düsseldorf, 40225 Düsseldorf, Germany b Department of Endocrinology, Diabetology, and Rheumatology, Heinrich Heine University Düsseldorf, 40225 Düsseldorf, Germany c Materials Science and Engineering, Yonsei University, Seoul 120-749, Korea article info Article history: Received 19 May 2010 Received in revised form 20 July 2010 Accepted 27 July 2010 Available online 6 August 2010 Keywords: Autodisplay 60-kDa Ro/SS-A antigen SLE ELISA SD–ELISA abstract Enzyme-linked immunosorbent assay (ELISA) is a common tool to test human sera on an antibody reac- tion against a specific antigen. The 60-kDa Ro/SS-A antigen for autoantibodies can be found in sera from systemic lupus erythematosus (SLE) patients. As in the case of 60-kDa Ro/SS-A, antigens used in ELISAs are recombinantly expressed in Escherichia coli and time-consuming purification steps are needed to get the proteins. To avoid these disadvantages, 60-kDa Ro/SS-A was expressed on the surface of E. coli using autodisplay, an efficient surface display system. Cells displaying 60-kDa Ro/SS-A on the surface were applied as an antigen source instead of the purified antigen. In total, 39 patients and 30 control sera were screened on a 60-kDa Ro/SS-A antibody reaction. To eliminate antibodies against native E. coli, human sera were preabsorbed with E. coli cells prior to the assay. The new ELISA protocol (surface display ELISA [SD–ELISA]) using E. coli with autodisplayed 60-kDa Ro/SS-A showed a sensitivity of 86.67% and a specificity of 83.33% by a cutoff value of 0.28. Our results show that autodisplay provides simple, rapid, and cheap access to human antigens for an ELISA to screen human sera against specific antibody reactions. Ó 2010 Elsevier Inc. All rights reserved. The 60-kDa Ro/SS-A antigen is a nuclear and cytoplasmic poly- peptide and consists of two domains. One domain contains a series of a-helical repeats arranged as a ring. The second domain adopts a Rossmann fold similar to that of the von Willebrand factor [1]. It could be shown that 60-kDa Ro/SS-A complexes with small RNAs known as Y RNA (Y for cYtoplasmic) [2]. Recent studies indicated that 60-kDa Ro/SS-A also binds misfolded small RNAs [3,4] and facilitates cell survival after ultraviolet exposure [5]. Antibodies against the Ro/SS-A cellular antigen are one of the most common types of autoantibodies detectable during routine immunological studies of autoimmune patients [6]. Anti-Ro/SS-A antibodies have been associated with an increasing number of rheumatic diseases. Common methods show that 35% of patients with systemic lupus erythematosus (SLE), 1 62% of patients with primary Sjögren’s syn- drome (pSS), 33% of patients with scleroderma, and 23% of patients with rheumatoid arthritis (RA) react positively [6–8]. A high anti- body concentration is associated with neonatal lupus or complete permanent congenital heart block [6,7]. Until now, most of the antigens for an enzyme-linked immuno- sorbent assay (ELISA), including human 60-kDa Ro/SS-A, were pre- pared by recombinant expression in Escherichia coli [9]. To finally obtain these proteins, cells must be lysed and several purification steps may be required. This means that the extraction of the anti- gen, if expression in E. coli was indeed successful, can be time-con- suming, laborious, and expensive. For a standard ELISA, a 96-well microplate is coated with the purified antigen and human sera are screened by a common protocol. In the current study, the dis- advantages of the purification steps were intended to be avoided by establishing a new protocol. For this purpose, the human 60- kDa Ro/SS-A antigen was expressed on the cell surface of E. coli using autodisplay. These cells were directly applied to coat a 96- well microplate, and subsequently human sera were screened. Two preabsorption steps were needed to prevent nonspecific 0003-2697/$ - see front matter Ó 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.ab.2010.07.030 ⇑ Corresponding author. Fax: +49 211 8113847. E-mail address: joachim.jose@uni-duesseldorf.de (J. Jose). 1 Abbreviations used: SLE, systemic lupus erythematosus; pSS, primary Sjögren’s syndrome; RA, rheumatoid arthritis; ELISA, enzyme-linked immunosorbent assay; SD–ELISA, surface display ELISA; IgG, immunoglobulin G; HRP, horseradish peroxi- dase; FITC, fluorescein isothiocyanate; TMB, 3,3 0 ,5,5 0 -tetramethylbenzidine; PCR, polymerase chain reaction; LB, lysogeny broth; EDTA, ethylenediaminetetraacetate; IPTG, isopropyl-b-D-thiogalactopyranoside; SDS, sodium dodecyl sulfate; DTT, dithi- othreitol; PAGE, polyacrylamide gel electrophoresis; PBS, phosphate-buffered saline; BSA, bovine serum albumin; RT, room temperature; FCS, fetal calf serum; ROC, receiver operating characteristics; AUC, area under the curve. Analytical Biochemistry 407 (2010) 72–78 Contents lists available at ScienceDirect Analytical Biochemistry journal homepage: www.elsevier.com/locate/yabio