Combining EL4-B5-based B-cell stimulation and phage display technology for the successful isolation of human anti-Scl-70 autoantibody fragments.
Scl-70 is the major antigen recognised by autoantibodies in the sera of patients with systemic sclerosis (SSc). The autoantibodies that specifically react with Scl-70 are highly characteristic of the disease and represent valuable markers for the diagnosis of SSc. We describe a novel strategy for cloning autoantibody fragments starting with a small blood sample from an SSc patient. B cells isolated from the collected peripheral blood mononuclear cells (PBMCs) were cultured in vitro using the EL4-B5 system. Anti-Scl-70 IgG-producing cells were pooled for RNA preparation followed by the generation of phagemid libraries of approximately 10(7) independent single-chain Fvs (scFvs). The screening of these libraries by phage display allowed us to isolate four anti-Scl-70 scFvs following three rounds of biopanning. About 10 times more starting blood material was needed to generate scFv libraries of similar size from PBMCs of an SSc patient and only two anti-Scl-70 scFvs were isolated after three rounds of phage selection. Together, this work shows that functional autoantibody fragments can be advantageously cloned after in vitro expansion of B cells. The isolated anti-Scl-70 autoantibody fragments represent useful tools for calibrating SSc diagnostic assays.
Weber
Malte
M
Department of Rare Reagents, Roche Diagnostics GmbH, Nonnenwald 2, Penzberg 82372, Germany.
Weiss
Etienne
E
Engel
Alfred M
AM
D001323:Autoantibodies / Q000302:isolation & purification*; D001402:B-Lymphocytes / Q000378:metabolism*; D002478:Cells, Cultured; D003001:Cloning, Molecular; D004264:DNA Topoisomerases, Type I; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D007128:Immunoglobulin Fragments / Q000302:isolation & purification; D007135:Immunoglobulin Variable Region / Q000235:genetics; D008213:Lymphocyte Activation; D009687:Nuclear Proteins / Q000276:immunology*; D019151:Peptide Library; D016133:Polymerase Chain Reaction; D012595:Scleroderma, Systemic / Q000175:diagnosis* / Q000276:immunology
D001323:Autoantibodies; D007128:Immunoglobulin Fragments; D007135:Immunoglobulin Variable Region; D009687:Nuclear Proteins; D019151:Peptide Library; C048100:Scl 70 antigen, human; D004264:DNA Topoisomerases, Type I