Fig 2: Test of CCL21-antibodies using Western blot analysis. The Western blot analysis showed that three of the CCL21 antibodies detected both full length and tailless CCL21 (four left lanes). The custom-made anti-C-terminal tail of CCL21 antibody from Sicgen only detected full length CCL21 (right lane).

Fig 3: CCL21 serum levels and correlation analysis in ELISA 1, Luminex and ELISA 2. (A) The mean serum levels of CCL21 in ELISA 1 were higher in SSc patients compared to healthy controls (i) in Luminex (ii) and ELISA 2 (iii). (B) In ELISA 1, CCL21 serum levels were higher in patients diagnosed with PAH compared to patients with no PH (i) while similar in PAH patient and no PH (ii) in Luminex. The mean serum levels of CCL21 in ELISA 2 however were, like in ELISA 1, higher in PAH patients compared to those without PH (iii). (C) The correlation of CCL21 serum levels between ELISA 1 and Luminex (r=0.515, p<0.001) (i) and the correlation of CCL21 serum levels between ELISA 2 and Luminex (r=0.52, p<0.001) (ii) were poor. However, the correlation of CCL21 serum levels between ELISA 2 and ELISA 1 (r=0.788, p<0.001) (iii) were good.

Fig 4: CCL21 levels in SSc, SS and MCTD of anti-CCL21 antibody positive and negative patients. (A) Mean serum levels of CCL21 in anti-CCL21 antibody positive (green) and anti-CCL21 antibody negative (red) patients in SSc, SS and MCTD. (B) Mean serum levels of CCL21 of anti-CCL21 antibody positive (green) and anti-CCL21 antibody negative (red) patients in SSc patients diagnosed with PAH.

Fig 5: ROC analysis of CCl21 levels from ELISA 1, Luminex and ELISA 2. (A) ROC analysis of CCL21 levels from ELISA 1 with AUC=0.843, 95% CI = 0.084-0.883 and p<0.001, (B) ROC analysis of CCL21 levels from Luminex with AUC=0.772, 95% CI = 0.711-0.833 and p<0.001, and (C) ROC analysis of CCL21 levels from ELISA 2 with AUC=0.770, 95% CI = 0.711-0.830 and p<0.001.