Anti-beta(2)GP-I and anti-prothrombin antibodies generated by phage display.
This chapter describes the construction and screening of a library of single-chain variable fragments (svFv) derived from patients with autoimmune disease. The methods cover the isolation of mononuclear cells from peripheral blood, preparation of RNA, and recovery of immunoglobulin-coding sequences by polymerase chain reaction (PCR). Cloning into a phage display vector and screening of the scFv display library by a simple panning procedure are described. These methods are applicable to library construction from any patient group or (with alternative primer sets) any mammalian species.
Cabiedes
Javier
J
Department of Immunology and Rheumatology, Instituto Nacional de Ciencias Médicas y Nutrición Salvador Zubirán, Tlalpan, Mexico City, 14000, Mexico. cabiedesj@yahoo.com
Cabral
Antonio R
AR
D000888:Antibodies, Anti-Idiotypic / Q000276:immunology*; D000911:Antibodies, Monoclonal / Q000235:genetics / Q000276:immunology; D001327:Autoimmune Diseases / Q000235:genetics / Q000276:immunology; D017931:DNA Primers / Q000737:chemistry; D005822:Genetic Vectors; D006801:Humans; D007143:Immunoglobulin Heavy Chains / Q000235:genetics / Q000276:immunology; D007147:Immunoglobulin Light Chains / Q000235:genetics / Q000276:immunology; D007135:Immunoglobulin Variable Region / Q000235:genetics / Q000276:immunology*; D019151:Peptide Library; D016133:Polymerase Chain Reaction / Q000379:methods; D011516:Prothrombin / Q000276:immunology*; D053482:beta 2-Glycoprotein I / Q000276:immunology*
D000888:Antibodies, Anti-Idiotypic; D000911:Antibodies, Monoclonal; D017931:DNA Primers; D007143:Immunoglobulin Heavy Chains; D007147:Immunoglobulin Light Chains; D007135:Immunoglobulin Variable Region; D019151:Peptide Library; D053482:beta 2-Glycoprotein I; D011516:Prothrombin