Anti-Ap^1-17 Abs from different SSc patients recognize different motifs within the same peptide.

<p>Microtiter plates were coated with 10 µg/ml anti-Ap^1-17 Abs from 8 SSc patients or with IVIG as negative control. Supernatants from pt4 (Panel A) and pt14 (Panel B) phage clones were diluted 16-fold in PBS and added to the wells (100 µl/well). After a 4-h incubation, the Ab-phage clone interaction was detected with HRP-conjugated anti-M13 mAb and <i>o</i>-phenylenediamine. Each data point is the mean of duplicate wells. The data are representative of two experiments.</p

Numbers of human proteins expressing the pt4 and pt14 antigenic motifs, by organ system (apparatus) and specific tissue.

a)<p>The number of proteins expressing the pt4 and pt14 motifs were determined by searching in the Swiss-Prot database (<a href="http://prosite.expasy.org/scanprosite/" target="_blank">http://prosite.expasy.org/scanprosite/</a>), using human tissue expression (<u>Bgee</u> data) filters.</p

Reactivity of affinity-purified anti-Ap^1-17 Abs from 3 SSc patients.

<p>(<b>A.</b>) Recombinant human CENP-B and CENP-A proteins were loaded on alternative lanes (200 ng/lane) of a 12.5% SDS mini-gel under non-reducing conditions, transferred to a PVDF filter, and incubated for 3 h with affinity-purified anti-Ap^1-17 Abs from pt1, pt4 and pt14. Serum from pt1 and IVIG were used as controls. Bound Abs were detected using HRP-conjugated goat anti-human IgG and diaminobenzidine substrate solution. (<b>B.</b>) Centromere staining of pt1, pt4, pt8 and pt14 anti-Ap^1-17 IgG and of their corresponding serum (1∶100 dilution) to fixed permeabilized HeLa cells. Bound IgG was revealed by fluorescence staining with FITC-conjugated anti-human IgG (Fc portion). Cells were examined with a Nikon confocal microscope and a CCD camera (Nikon digital sight DS-U1), using a 60× Plan Apo VC objective.</p

Specific peptide inhibition of phage particle binding to anti-Ap^1-17 IgG demonstrates that phage insert sequences recognize the antigen-combining sites of anti-Ap^1-17 IgG.

<p>Microtiter plates were coated with affinity-purified anti-Ap^1-17 IgG from pt4 (A.) or pt14 (B.). After the blocking of free protein-binding sites with PBS-BSA, wells were incubated for 2 h with 50 µl PBS containing serial dilutions of either free (closed symbols) or KLH-conjugated (open symbols) Ap^1-17 (rhombus), Ap^17-30 (triangle), CBp^1-13 (circle) or Qp-1a (square). Then, without removing the inhibitor, 50 µl/well of an appropriate dilution of phage supernatant pc4.33 (A.) and pc14.49 (B.) was added and incubation prolonged for 2 h. Bound phage particles were detected by sequential addition of HRP-anti-M13 mAb and o-phenylenediamine solution. Results are expressed as percentage of binding inhibition. Each data point is the mean of duplicate wells. The data are representative of two experiments. The maximum amount of inhibitor (800 µg) corresponds to 382 nmol Ap^1-17, 558 nmol Ap^17-30, 486 nmol CBp^1-13 and 574 nmol Qp-1a.</p

Specificity of anti-Ap^1-17 IgG for CENP-A documented by human recombinant CENP-A inhibition of anti-Ap^1-17 IgG binding to KLH-Ap^1-17.

<p>Anti-Ap^1-17 Abs from pt4 (A) and pt14 (B) were diluted in PBS-T20 at the lowest concentration giving 80%–100% of maximal A₄₉₀ in binding assay, and pre-incubated with an equal volume of PBS containing 2-fold serial dilutions of CENP-A (○), CENP-B (•), and CENP-B-derived peptide CBp^1-13 (▪). Following a 2-h incubation, the mixture was added to microtiter plate wells coated with KLH-Ap^1-17. After a 4-h incubation and three washes, bound IgG was detected with HRP-conjugated anti-human IgG (Fc portion) and <i>o</i>-phenylenediamine. Inhibition by Ap^1-17 peptide (□) and by Qp-1a (X) were included as positive and negative controls, respectively. Results are expressed as percentage of binding inhibition. The data are representative of 2 experiments.</p

Screening of SSc sera for specificity to Ap^1-17.

<p>(<b>A.</b>) Sera from SSc patients (30 ACA⁺, 20 Scl70⁺; 7 ACA⁻/Scl70⁻) were screened for specificity to Ap^1-17 peptide in an indirect ELISA. Microtiter plates were coated with 5 µg/ml KLH-conjugated Ap^1-17 (filled circles) or KLH-conjugated Qp1-a (open circles). Wells were incubated for 4 h with serum samples (diluted 1∶100) from the three groups of patients and from 10 healthy blood donors (HBD); samples were tested in duplicate. Bound IgG was revealed with HRP-conjugated anti-human IgG (Fc portion) and <i>o</i>-phenylenediamine. Each data point is the mean of duplicate wells (SEM ≤8%). Horizontal lines indicate the mean for each group. O.D., optical density. *Mann-Whitney p<0.0001. (<b>B</b>) ROC analysis was performed by including the absorbance binding of each patient's serum to Ap^1-17 (minus the absorbance of the same sera to the unrelated peptide Qp1-a) as variable, and by comparing the ACA⁺ group to control ACA⁻ group.</p

Alignment of pt4 and pt14 deduced motifs with CENP-A protein sequence.

<p>Motif amino acid “S” shown in small letter could be used interchangeably with “T”.</p