Fig 1.
Electrophoretic mobility of carbamylated serum proteins and their immunoreactivity with anti-CarP Ab.
(A) Native and carbamylated proteins (2 μg per lane) were analyzed by 10% SDS-PAGE under reducing conditions and stained with Coomassie brilliant blue. Molecular weight markers were run on a parallel track. (B) Native and carbamylated proteins (500 ng/lane) were separated by 10% SDS-PAGE under reducing conditions, transferred to a PVDF filter, and incubated overnight with affinity-purified anti-CarP Ab. Bound Ab were detected using HRP-conjugated goat anti-human IgG and ECL substrate. BSA, bovine serum albumin; HA, human albumin; FIB, fibrinogen; Car, carbamylated.
Fig 2.
Specificity of anti-CarP Ab assessed by binding and inhibition ELISAs.
(A) In the binding ELISA, microtiter plates were coated with BSA (square), HA (circle), or FIB (triangle), in carbamylated form (empty symbols) or native form (filled symbols). Wells were incubated PBS containing serial dilutions of anti-CarP Ab. Standard (STD) serum was used as specificity control. Bound IgG was revealed with HRP-conjugated anti-human IgG and o-phenylenediamine. The data are representative of at least two independent experiments. (B) In the inhibition assay, anti-CarP Ab were diluted in PBS-BSA to the lowest concentration giving 80%–100% of maximal A490 in the binding assay (2.5 μg/ml), and pre-incubated with an equal volume of PBS containing 2-fold serial dilutions of BSA (square), HA (circle), or FIB (triangle), in carbamylated form (empty symbols) or native form (filled symbols). Following a 2-h incubation at room temperature, the mixture was added to microtiter plate wells coated with CarBSA. After a 4-h incubation at room temperature and three washes, bound IgG was detected with HRP-conjugated anti-human IgG (Fc portion) and o-phenylenediamine. Results are expressed as percentage of binding inhibition compared with binding in the absence of inhibitor. The data are representative of at least two independent experiments.
Table 1.
Characteristics of the 124 patients with systemic sclerosis included in the study.
Table 2.
Disease severity scores in patients with systemic sclerosis.
Fig 3.
Levels of anti-CarP Ab in SSc patients and healthy blood donors.
(A) Sera from 124 SSc patients and 41 healthy blood donors were screened for specificity to CarBSA by indirect ELISA. Binding of anti-CarP Ab is expressed as a percentage of the binding obtained with positive control serum from an RA patient. (B) Anti-CarP Ab levels in SSc patients divided by age at observation into five groups: I, ≤44 years, 32 cases; II, 44–53 years, 28 cases; III, 54–59 years, 24 cases; IV, 60–66 years, 17 cases; and V, ≥67 years, 23 cases. Horizontal bars mark the medians and boxes indicate interquartile ranges; outliers (more than 1.5 times the interquartile range) are marked with circles, while extreme outliers (more than 3 times the interquartile range) are marked with asterisks. Kruskal-Wallis Htest, *p = 0.037, **p = 0.014.
Table 3.
Antibodies (Ab) to carbamylated BSA (CarBSA) inversely correlate with age and modified Rodnan skin score in patients with systemic sclerosis.
Fig 4.
Receiver operating characteristic (ROC) analysis to define levels of anti-CarP Ab (cut-offs) that distinguish patients according to clinical variables.
(A) SSc patients with mRss>0 vs those with mRss = 0. (B) SSc patients with scleredema vs those without it.
Table 4.
Antibodies (Ab) to carbamylated BSA (CarBSA) are associated to a worse skin involvement.