Identification of Rat Targets of Anti-Soluble Liver Antigen Autoantibodies by Serologic Proteome Analysis

Abstract

Background: Anti-soluble liver antigen (SLA) autoantibodies are specific for autoimmune hepatitis type 1 and are the only immunologic marker found in 15-20% of hepatitis cases previously considered cryptogenic. Anti-SLA antibodies react with the 100 000g supernatant from rat liver homogenate, but the molecular targets remain controversial. Methods: We characterized anti-SLA targets by oneand two-dimensional immunoblotting analysis. The recognized proteins were identified by peptide mass fingerprint analysis after matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. Results: Three proteins of 35 kDa and pI 6.0, 50 kDa and pI between 6.0 and 6.5, and 58 kDa and pI between 6.5 and 7.0 were stained more intensely by anti-SLA positive-sera than by control sera. After in-gel tryptic digestion, MALDI-TOF analysis of the generated peptides enabled the clear identification of N-hydroxyarylamine sulfotransferase, isoforms of ␣-enolase, and isoforms of catalase. Conclusions: Possible antigens for anti-SLA antibodies include a sulfotransferase, ␣-enolase(s), and catalase(s). Two-dimensional electrophoresis combined with mass spectrometry offers a versatile tool to identify molecular targets of autoantibodies and thus to improve diagnostic tools and the understanding of the immune process.

Identification of In Vivo-Expressed Immunogenic Proteins by Serological Proteome Analysis of the Bacillus anthracis Secretome

“…To identify such proteins in the complex proteome of a pathogen, assessment of seroreactivity can be combined with proteomic studies for direct selection of proteins that are able to elicit a humoral immune response in the course of bacterial infections. This approach, in which large numbers of proteins separated by two-dimensional electrophoresis (2-DE) are identified by mass spectrometry and probed with immune sera (designated serological proteome analysis [SERPA] [43]), has been used with several bacterial systems for identification of diagnostic markers or vaccine candidates (6,15,20,33,35,36,46,80,82,83) or for determination of specific circulating antibodies and/or disease markers in a variety of pathological states not related to diseases caused by bacteria (7,43).…”

Autoimmune liver serology: Current diagnostic and clinical challenges

“…Progress has been made in the definition of other autoantibodies frequently present in AIH but undetectable by IFL including antibodies against SLA/LP [51,[81][82][83][84][85] and ASGPR. Most of anti-SLA/LP positive patients are also positive for ANA, SMA or anti-LKM1, but occasionally anti-SLA is present in isolation and, in this case, its detection is of diagnostic importance [81,86] .…”

Immunoproteomics

“…Nonetheless, epitope prediction remains a popular first screening method to identify candidate T cell determinants for subsequent biological validation (78 -89), and predictive algorithms are frequently combined with in vitro MHC-binding assays to confirm experimentally that the predicted ligands bind to the targeted MHC molecule (80,90). A more comprehensive approach that allows assessment of natural processing and presentation of candidate epitopes involves the direct biochemical analysis of class I or class II ligands, which has been coined as the immunoproteome (82,83,(91)(92)(93)(94)(95)(96)(97)(98)(99).…”