In contrast, up to 74% of the PBC sera reacted with the two peptides 25 and 29 within the catalytic domain and the peptide 7 (aa 101-125) in the selleckchem first hinge region (Tables (Tables22 and and3),3), and IgG and IgM reactivity towards these three peptides was significantly higher in PBC patients than in controls (Figure (Figure22). Table 2 Reactivity and incidence of IgG-antibodies to 33 PDC-E2 peptides in PBC patients as compared to healthy individuals n (%) Figure 2 Box plots showing the reactivity of sera from 95 primary biliary cirrhosis (PBC) patients (grey bars) and 22 blood donors (white bars) with 33 overlapping peptides spanning the whole PDC-E2 sequence. IgG antibody reactivities. A: Peptide 1-16; B: Peptide …
Table 3 Reactivity and incidence of IgM-antibodies to 33 PDC-E2 peptides in PBC patients as compared to healthy individuals n (%) The peptides 15, 21 and 24 were the only ones whose reactivity was lower with PBC sera as compared to control sera (Figure (Figure2A,2A, ,BB and andDD). Most PBC sera recognized several peptides in parallel. IgM-antibodies generally reacted with a higher diversity of peptides than IgG-antibodies (IgG: mean + SD: 5.3 �� 7.7 peptides, median: 3 peptides, range: 0-30 peptides; IgM: mean �� SD: 9 �� 7.1 peptides, median: 7 peptides, range: 0-28 peptides). Two PBC sera had neither IgG- nor IgM-antibodies to any of the 33 peptides although both sera showed high antibody reactivities towards PDC and M2 in the ELISA. Sera from patients with AMA negative/ANA positive PBC hardly reacted with any of the peptides (Tables (Tables22 and and33).
Incidence and reactivity of antibodies to the different peptides in sera from patients with autoimmune hepatitis, alcoholic liver disease and collagen disorders resembled that in healthy controls (data not shown). Reactivity of PBC-sera with the inner lipoyl domain epitope aa 167-184 using different conjugates In view of the fact that the 25 mers peptide 10 (aa 152-176) and 11 (aa 169-193) used in the epitope mapping above did not completely respond to the published sequence 167-184 (AEIETDKATIGFEVQEEG), we also synthesized the latter peptide in an unlipoylated form and a form containing lipoic acid (LA) at aa K173 (peptide 167-184-LA) and applied both forms in the ELISA. In accordance to a previous study, also with these peptides reactivity of the 95 PBC sera was low (Table (Table44).
Table 4 Reactivity of sera from 95 anti-M2 positive PBC patients with different peptide conjugates containing the reported immunodominant epitope of PDC-E2[6,12] In order to find out whether the Brefeldin_A antibodies may recognize a conformational epitope the peptides were coupled to ovalbumin (OVA-peptide 167-184 and OVA-peptide 167-184-LA). With these conjugated peptides reactivity was stronger, but most sera showed a high reactivity already with OVA alone (Figure (Figure3).3).