Synthetic Peptide-Based ELISA and ELISpot Assay for Identifying Autoantibody Epitopes.
Enzyme-linked immunosorbent assay (ELISA) is an invaluable diagnostic tool to detect serum autoantibody binding to target antigen. To map the autoantigenic epitope(s), overlapping synthetic peptides covering the total sequence of a protein antigen are used. A large set of peptides synthesized on the crown of pins can be tested by Multipin ELISA for fast screening. Next, to validate the results, the candidate epitope peptides are resynthesized by solid-phase synthesis, coupled to ELISA plate directly, or in a biotinylated form, bound to neutravidin-coated surface and the binding of autoantibodies from patients' sera is tested by indirect ELISA. Further, selected epitope peptides can be applied in enzyme-linked immunospot assay to distinguish individual, citrullinated peptide-specific autoreactive B cells in a pre-stimulated culture of patients' lymphocytes.
Department of Immunology, Eötvös Loránd University, Pázmány Péter sétány 1/c, Budapest, 1117, Hungary.
Judit
J
Pozsgay
Department of Immunology, Eötvös Loránd University, Pázmány Péter sétány 1/c, Budapest, 1117, Hungary.
Eszter
E
Szarka
Department of Immunology, Eötvös Loránd University, Pázmány Péter sétány 1/c, Budapest, 1117, Hungary.
Krisztina
K
Huber
MTA-ELTE Research Group of Peptide Chemistry, Hungarian Academy of Sciences, Budapest, Hungary.
Fruzsina
F
Babos
MTA-ELTE Research Group of Peptide Chemistry, Hungarian Academy of Sciences, Budapest, Hungary.
Anna
A
Magyar
Department of Organic Chemistry, Eötvös Loránd University, Budapest, Hungary.|MTA-ELTE Research Group of Peptide Chemistry, Hungarian Academy of Sciences, Budapest, Hungary.
Ferenc
F
Hudecz
Department of Immunology, Eötvös Loránd University, Pázmány Péter sétány 1/c, Budapest, 1117, Hungary. sarmayg@elte.hu.
Gabriella
G
Sarmay
D001120:Arginine; D001323:Autoantibodies / Q000276:immunology*; D001360:Avidin / Q000378:metabolism; D001402:B-Lymphocytes / Q000276:immunology; D002469:Cell Separation; D002956:Citrulline / Q000378:metabolism; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D058501:Enzyme-Linked Immunospot Assay / Q000379:methods*; D018604:Epitope Mapping / Q000379:methods*; D006801:Humans; D010455:Peptides / Q000138:chemical synthesis* / Q000737:chemistry / Q000276:immunology* / Q000378:metabolism
D001323:Autoantibodies; D010455:Peptides; C093483:neutravidin; D001360:Avidin; D002956:Citrulline; D001120:Arginine
Anti-citrullinated peptide antibodies; B cell; Citrulline-peptide; Diagnosis; ELISA; ELISpot; Multipin ELISA; Rheumatoid arthritis