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Affinity purified anti-citrullinated protein/peptide antibodies target antigens expressed in the rheumatoid joint
INTRODUCTION: A major subset of patients with rheumatoid arthritis (RA) is characterized by the presence of circulating autoantibodies directed to citrullinated proteins/peptides (ACPAs). These autoantibodies, which are commonly detected by using an enzyme-linked immunosorbent assay (ELISA) based on...
Autores principales: | , , , , , , , , , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
BioMed Central
2014
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4448322/ https://www.ncbi.nlm.nih.gov/pubmed/25112157 http://dx.doi.org/10.1186/ar4683 |
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author | Ossipova, Elena Cerqueira, Cátia Fernandes Reed, Evan Kharlamova, Nastya Israelsson, Lena Holmdahl, Rikard Nandakumar, Kutty Selva Engström, Marianne Harre, Ulrike Schett, Georg Catrina, Anca I Malmström, Vivianne Sommarin, Yngve Klareskog, Lars Jakobsson, Per-Johan Lundberg, Karin |
author_facet | Ossipova, Elena Cerqueira, Cátia Fernandes Reed, Evan Kharlamova, Nastya Israelsson, Lena Holmdahl, Rikard Nandakumar, Kutty Selva Engström, Marianne Harre, Ulrike Schett, Georg Catrina, Anca I Malmström, Vivianne Sommarin, Yngve Klareskog, Lars Jakobsson, Per-Johan Lundberg, Karin |
author_sort | Ossipova, Elena |
collection | PubMed |
description | INTRODUCTION: A major subset of patients with rheumatoid arthritis (RA) is characterized by the presence of circulating autoantibodies directed to citrullinated proteins/peptides (ACPAs). These autoantibodies, which are commonly detected by using an enzyme-linked immunosorbent assay (ELISA) based on synthetic cyclic citrullinated peptides (CCPs), predict clinical onset and a destructive disease course. In the present study, we have used plasma and synovial fluids from patients with RA, for the affinity purification and characterization of anti-CCP2 reactive antibodies, with an aim to generate molecular tools that can be used in vitro and in vivo for future investigations into the pathobiology of the ACPA response. Specifically, this study aims to demonstrate that the surrogate marker CCP2 can capture ACPAs that bind to autoantigens expressed in vivo in the major inflammatory lesions of RA (that is, in the rheumatoid joint). METHODS: Plasma (n = 16) and synovial fluid (n = 26) samples were collected from RA patients with anti-CCP2 IgG levels of above 300 AU/mL. Total IgG was isolated on Protein G columns and subsequently applied to CCP2 affinity columns. Purified anti-CCP2 IgG was analyzed for reactivity and specificity by using the CCPlus® ELISA, in-house peptide ELISAs, Western blot, and immunohisto-/immunocytochemistry. RESULTS: Approximately 2% of the total IgG pool in both plasma and synovial fluid was CCP2-reactive. Purified anti-CCP2 reactive antibodies from different patients showed differences in binding to CCP2 and differences in binding to citrullinated peptides from α-enolase, vimentin, fibrinogen, and collagen type II, illustrating different ACPA fine-specificity profiles. Furthermore, the purified ACPA bound not only in vitro citrullinated proteins but, more importantly, in vivo-generated epitopes on synovial fluid cells and synovial tissues from patients with RA. CONCLUSIONS: We have isolated ACPAs from plasma and synovial fluid and demonstrated that the CCP2 peptides, frequently used in diagnostic ELISAs, de facto act as surrogate antigens for at least four different, well-characterized, largely non-cross-reactive, ACPA fine specificities. Moreover, we have determined the concentration and proportion of CCP2-reactive IgG molecules in rheumatoid plasma and synovial fluid, and we have shown that the purified ACPAs can be used to detect both in vitro- and in vivo-generated citrullinated epitopes by various techniques. We anticipate that these antibodies will provide us with new opportunities to investigate the potential pathogenic effects of human ACPAs. |
format | Online Article Text |
id | pubmed-4448322 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2014 |
publisher | BioMed Central |
record_format | MEDLINE/PubMed |
spelling | pubmed-44483222015-05-30 Affinity purified anti-citrullinated protein/peptide antibodies target antigens expressed in the rheumatoid joint Ossipova, Elena Cerqueira, Cátia Fernandes Reed, Evan Kharlamova, Nastya Israelsson, Lena Holmdahl, Rikard Nandakumar, Kutty Selva Engström, Marianne Harre, Ulrike Schett, Georg Catrina, Anca I Malmström, Vivianne Sommarin, Yngve Klareskog, Lars Jakobsson, Per-Johan Lundberg, Karin Arthritis Res Ther Research Article INTRODUCTION: A major subset of patients with rheumatoid arthritis (RA) is characterized by the presence of circulating autoantibodies directed to citrullinated proteins/peptides (ACPAs). These autoantibodies, which are commonly detected by using an enzyme-linked immunosorbent assay (ELISA) based on synthetic cyclic citrullinated peptides (CCPs), predict clinical onset and a destructive disease course. In the present study, we have used plasma and synovial fluids from patients with RA, for the affinity purification and characterization of anti-CCP2 reactive antibodies, with an aim to generate molecular tools that can be used in vitro and in vivo for future investigations into the pathobiology of the ACPA response. Specifically, this study aims to demonstrate that the surrogate marker CCP2 can capture ACPAs that bind to autoantigens expressed in vivo in the major inflammatory lesions of RA (that is, in the rheumatoid joint). METHODS: Plasma (n = 16) and synovial fluid (n = 26) samples were collected from RA patients with anti-CCP2 IgG levels of above 300 AU/mL. Total IgG was isolated on Protein G columns and subsequently applied to CCP2 affinity columns. Purified anti-CCP2 IgG was analyzed for reactivity and specificity by using the CCPlus® ELISA, in-house peptide ELISAs, Western blot, and immunohisto-/immunocytochemistry. RESULTS: Approximately 2% of the total IgG pool in both plasma and synovial fluid was CCP2-reactive. Purified anti-CCP2 reactive antibodies from different patients showed differences in binding to CCP2 and differences in binding to citrullinated peptides from α-enolase, vimentin, fibrinogen, and collagen type II, illustrating different ACPA fine-specificity profiles. Furthermore, the purified ACPA bound not only in vitro citrullinated proteins but, more importantly, in vivo-generated epitopes on synovial fluid cells and synovial tissues from patients with RA. CONCLUSIONS: We have isolated ACPAs from plasma and synovial fluid and demonstrated that the CCP2 peptides, frequently used in diagnostic ELISAs, de facto act as surrogate antigens for at least four different, well-characterized, largely non-cross-reactive, ACPA fine specificities. Moreover, we have determined the concentration and proportion of CCP2-reactive IgG molecules in rheumatoid plasma and synovial fluid, and we have shown that the purified ACPAs can be used to detect both in vitro- and in vivo-generated citrullinated epitopes by various techniques. We anticipate that these antibodies will provide us with new opportunities to investigate the potential pathogenic effects of human ACPAs. BioMed Central 2014-08-12 2014 /pmc/articles/PMC4448322/ /pubmed/25112157 http://dx.doi.org/10.1186/ar4683 Text en © Ossipova et al.; licensee BioMed Central Ltd. 2014 This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. |
spellingShingle | Research Article Ossipova, Elena Cerqueira, Cátia Fernandes Reed, Evan Kharlamova, Nastya Israelsson, Lena Holmdahl, Rikard Nandakumar, Kutty Selva Engström, Marianne Harre, Ulrike Schett, Georg Catrina, Anca I Malmström, Vivianne Sommarin, Yngve Klareskog, Lars Jakobsson, Per-Johan Lundberg, Karin Affinity purified anti-citrullinated protein/peptide antibodies target antigens expressed in the rheumatoid joint |
title | Affinity purified anti-citrullinated protein/peptide antibodies target antigens expressed in the rheumatoid joint |
title_full | Affinity purified anti-citrullinated protein/peptide antibodies target antigens expressed in the rheumatoid joint |
title_fullStr | Affinity purified anti-citrullinated protein/peptide antibodies target antigens expressed in the rheumatoid joint |
title_full_unstemmed | Affinity purified anti-citrullinated protein/peptide antibodies target antigens expressed in the rheumatoid joint |
title_short | Affinity purified anti-citrullinated protein/peptide antibodies target antigens expressed in the rheumatoid joint |
title_sort | affinity purified anti-citrullinated protein/peptide antibodies target antigens expressed in the rheumatoid joint |
topic | Research Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4448322/ https://www.ncbi.nlm.nih.gov/pubmed/25112157 http://dx.doi.org/10.1186/ar4683 |
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