Immunoglobulin G structure and rheumatoid factor epitopes

Antibodies are important for immunity and exist in several classes (IgM, IgD, IgA, IgG, IgE). They are composed of symmetric dimeric molecules with two antigen binding regions (Fab) and a constant part (Fc), usually depicted as Y-shaped molecules. Rheumatoid factors found in patients with rheumatoid arthritis are autoantibodies binding to IgG and paradoxically appear to circulate in blood alongside with their antigen (IgG) without reacting with it. Here, it is shown that rheumatoid factors do not react with native IgG in solution, and that their epitopes only become accessible upon certain physico-chemical treatments (e.g. heat treatment at 57 °C), by physical adsorption on a hydrophobic surface or by antigen binding. Moreover, chemical cross-linking in combination with mass spectrometry showed that the native state of IgG is a compact (closed) form and that the Fab parts of IgG shield the Fc region and thereby control access of rheumatoid factors and presumably also some effector functions. It can be inferred that antibody binding to pathogen surfaces induces a conformational change, which exposes the Fc part with its effector sites and rheumatoid factor epitopes. This has strong implications for understanding antibody structure and physiology and necessitates a conceptual reformulation of IgG models

Physical Characteristics of a Citrullinated Pro-Filaggrin Epitope Recognized by Anti-Citrullinated Protein Antibodies in Rheumatoid Arthritis Sera.

Rheumatoid arthritis (RA) is an autoimmune disease of complex etiology. A characteristic feature of a subset of RA is the presence of anti-citrullinated protein antibodies (ACPA), which correlate with a progressive disease course. In this study, we employed streptavidin capture enzyme-linked immunosorbent assay to analyze ACPA reactivity. Using the pro-filaggrin peptide HQCHQEST-Cit-GRSRGRCGRSGS, as template, we analyzed the reactivity of RA sera and healthy donor sera to various peptides in order to determine the physical characteristics of the citrullinated pro-filaggrin epitope and to examine whether biotin labelling influence antibody recognition. The full-length cyclic pro-filaggrin peptide and a linear form with a N-terminal biotin, was recognized to the same level, whereas, a notable difference in ACPA reactivity to the linear peptides with a C-terminal biotin was found, probably due to steric hindrance. Screening of linear and cyclic truncated peptides, revealed that small cyclic peptides containing 10-12 amino acids are favored over the linear. Moreover, the charged amino acids C-terminal to citrulline were found to be essential for antibody reactivity, most important was the charged amino acid in position 4 C-terminal to citrulline. Collectively, peptide structure, length, the presence of charged amino acids and biotin labelling markedly influence antibody reactivity. In relation to the clinical diagnostics of ACPA, these findings may reflect the differences in diagnostic assays used for detection of ACPA, which relates to differences in sensitivity and specificity dependent on the assay applied

Application of synthetic peptides for detection of anti-citrullinated peptide antibodies

Anti-citrullinated protein antibodies (ACPAs) are a hallmark of rheumatoid arthritis (RA) and represent an important tool for the serological diagnosis of RA. In this study, we describe ACPA reactivity to overlapping citrullinated Epstein-Barr virus nuclear antigen-1 (EBNA-1)-derived peptides and analyze their potential as substrates for ACPA detection by streptavidin capture enzyme-linked immunosorbent assay. Using systematically overlapping peptides, containing a 10 amino acid overlap, labelled with biotin C-terminally or N-terminally, sera from 160 individuals (RA sera (n=60), healthy controls (n=40), systemic lupus erythematosus (n=20), Sjögren's syndrome (n=40)) were screened for antibody reactivity. Antibodies to a panel of five citrullinated EBNA-1 peptides were found in 67% of RA sera, exclusively of the IgG isotype, while 53% of the patient sera reacted with a single peptide, ARGGSRERARGRGRG-Cit-GEKR, accounting for more than half of the ACPA reactivity alone. Moreover, these antibodies were detected in 10% of CCP2-negative RA sera. In addition, 47% of the RA sera reacted with two or three citrullinated EBNA-1 peptides from the selected peptide panel. Furthermore, a negative correlation between the biotin attachment site and the location of citrulline in the peptides was found, i.e. the closer the citrulline was located to biotin, the lower the antibody reactivity. Our data suggest that citrullinated EBNA-1 peptides may be considered a substrate for the detection of ACPAs and that the presence of Epstein-Barr virus may play a role in the induction of these autoantibodies

Un jour/Un document - Les ouvriers ont-ils tous la même "valeur"? La hiérarchisation des ouvriers en fonction de leur nationalité

Dans le cadre des travaux du Haut Comité consultatif de la Population et de la Famille, préparation d'un "Projet pour un plan de l'immigration étrangère" par Georges Mauco en 1945. Ce projet implique une importante sélection ethnique. Ce tableau sur la valeur des ouvriers selon leur nationalité est annexé au projet. Il utilise différents indicateurs (sans que l'on sache comment ils sont construits) pour hiérarchiser les ouvriers en fonction de leur aspect physique, de leur régularité au trava..

Reactivity of RA sera (n = 20) to lysine-substituted linear citrullinated pro-filaggrin peptides analyzed by streptavidin capture ELISA.

<p>The citrullinated peptide HQCHQEST-Cit-GRSRGRCGRSGS was used as template. LCPa was used as positive control.</p

Pro-filaggrin peptides applied for reactivity screening

<p>Pro-filaggrin peptides applied for reactivity screening</p

Reactivity of RA sera (n = 20) and healthy donor sera (n = 20) to linear and cyclic citrullinated peptides pro-filaggrin peptides analyzed by streptavidin capture ELISA.

<p>The peptide HQCHQEST-Cit-GRSRGRCGRSGS was used as template, whereas the non-citrullinated peptide was used as control. B-LCPa and LCPa-B represent peptides with a N- and C-terminal biotin labelling, respectively. A. Reactivity of RA sera to pro-filaggrin peptides. B. Reactivity of healthy donor sera to pro-filaggrin peptides.</p

Reactivity of RA sera (n = 20) and healthy donor sera (n = 20) to linear and cyclic citrullinated pro-filaggrin peptides analyzed by streptavidin capture ELISA.

<p>The peptide HQCHQEST-Cit-GRSRGRCGRSGS was used as template, whereas the non-citrullinated peptide was used as control. A. Reactivity of RA sera to N-terminal biotinylated linear peptides. B. Reactivity of healthy donor sera to N-terminal biotinylated linear pro-filaggrin peptides. C. Reactivity of RA sera to C-terminal biotinylated linear peptides. D. Reactivity of healthy donor sera to C-terminal biotinylated linear pro-filaggrin peptides. E. Reactivity of RA sera to cyclic peptides. F. Reactivity of healthy donor sera to cyclic pro-filaggrin peptides.</p

The use of synthetic peptides for detection of anti-citrullinated protein antibodies in rheumatoid arthritis

Rheumatoid arthritis (RA) is an autoimmune disease of unknown etiology. A characteristic feature of RA is the presence of anti-citrullinated protein antibodies (ACPA). Since ACPAs are highly specific for RA and are often present before the onset of RA symptoms, they have become valuable diagnostic and prognostic. As a result, several assays for detection of ACPAs exist, which vary in sensitivity and specificity. In this study, we analyzed the reactivity of RA sera to selected peptides by solid-phase immunoassays in order to develop an ACPA assay with improved sensitivity and specificity. ACPA levels were determined with respect to sensitivity and specificity in 332 serum samples using the newly developed peptide panel, which was compared to the commercial assays CCPlus (Eurodiagnostica) and CCP3.1 (Inova Diagnostics).A primary panel (peptides 814, 33062 and 33156) was identified, which obtained a sensitivity of 71%, while the complete peptide panel reacted with 79% of RA sera screened. Total specificities of 89% and 80% were obtained for the primary peptide panel and the complete peptide panel. Sensitivities for the commercial assays ranged between 71% and 76% and specificities between 88% and 90%. These findings indicate that the generated peptide panel is optimal for ACPA detection and able to compete with commercial available assays. Collectively, this study may contribute to characterize autoimmunity towards citrullinated proteins and to the development of new and improved diagnostic assays for detection of ACPA and determination of RA