Affinity Purification and Comparative Biosensor Analysis of Citrulline-Peptide-Specific Antibodies in Rheumatoid Arthritis

Abstract

Background: In rheumatoid arthritis (RA), anti-citrullinated protein/peptide antibodies (ACPAs) are responsible for disease onset and progression, however, our knowledge is limited on ligand binding affinities of autoantibodies with different citrulline-peptide specificity.

Methods: Citrulline-peptide-specific ACPA IgGs were affinity purified and tested by ELISA. Binding affinities of ACPA IgGs and serum antibodies were compared by surface plasmon resonance (SPR) analysis. Bifunctional nanoparticles harboring a multi-epitope citrulline-peptide and a complement-activating peptide were used to induce selective depletion of ACPA-producing B cells.

Results: K_D values of affinity-purified ACPA IgGs varied between 10^-6 and 10^-8 M and inversely correlated with disease activity. Based on their cross-reaction with citrulline-peptides, we designed a novel multi-epitope peptide, containing Cit-Gly and Ala-Cit motifs in two-two copies, separated with a short, neutral spacer. This peptide detected antibodies in RA sera with 66% sensitivity and 98% specificity in ELISA and was recognized by 90% of RA sera, while none of the healthy samples in SPR. When coupled to nanoparticles, the multi-epitope peptide specifically targeted and depleted ACPA-producing B cells ex vivo.

Conclusions: The unique multi-epitope peptide designed based on ACPA cross-reactivity might be suitable to develop better diagnostics and novel therapies for RA.

Keywords: affinity; autoantibody; citrulline-peptides; cross-reaction; rheumatoid arthritis; targeting.

Figure 1

Recognition of citrulline-containing EBNA-2 and α-enolase peptide epitopes by sera of patients (RA, n = 177) and healthy volunteers (H, n = 104): (a,c) OD index: OD with Cit-peptide/OD with Arg peptide; and (b,d) receiver operating (ROC) curves. *** p ≤ 0.001. RA: rheumatoid arthritis.

Figure 2

Reactivity of affinity-purified IgG fractions with the relevant and irrelevant citrulline-containing peptides. OD ratios that are OD with Cit/OD with Arg-containing peptides are shown. Cut-off values for each Cit- and Arg-containing peptide pair were calculated from OD indexes of 120 healthy samples (the means of OD indexes + 2 × SD). These were below 1.5 for all peptides. Results of a typical experiment.

Figure 3

The Cit-multi-epitope peptide identifies RA sera with the highest specificity and 66% sensitivity: (a), ELISA, OD indexes (OD with Cit-peptide/OD with Arg-peptid) of RA (n = 210) and healthy (n = 90) samples, *** p ≤ 0.001; and (b) ROC curve of ELISA. Area under the curve (AUC): 0.7843.

Figure 4

Representative sensograms of: (a) Cit-filaggrin19-affinity purified IgG; (b) negative control IVIG; and two typical sera with: (c) higher; and (d) lower affinity on Cit-filaggrin19 coupled GLH chip.

Figure 5

Distribution of apparent K_D values of serum antibodies as measured from RA samples on GLH chip (top); and isoaffinity diagrams (bottom).

Figure 6

Distribution of apparent K_D values measured from RA sera on biotinylated Cit-collagen II, Cit-fibrin β and multi-epitope peptides immobilized on NLC chip.

Figure 7

Correlation between the disease severity (DAS28) and the apparent binding affinity (K_D) of serum antibodies from RA patients, (a) measured on filaggrin 19 peptide, (b) on fibrin β peptide.

Figure 8

Ex vivo inhibition of the Cit-peptide-specific antibody production by bifunctional PLGA nanoparticles covalently coupled to multi-epitope peptide and a complement-activating peptide. Antibody synthesis was tested by ELISpot assay according to a modified procedure of the Manufacturer (Mabtech, Stockholm, Sweden). As complement source, 1% normal human sera (NHS) was used; for the negative control, the sera were heat-inactivated (iNHS). (a) Average number of peptide-specific antibody-secreting cells (ELISpots) from 10⁶ PBMC of eight RA patients; *** p ≤ 0.001; and (b) ELISpot data of individual RA patients.