Vafa O, Sullivan KF. 3C17 (peptide A) and 25C38 (peptide B). Peptides A and B were reactive to 78 (86%) and 79 (87%) of ACA, respectively. Core antigens of hepatitis B disease (HBV) and hepatitis C disease (HCV) have related sequences to peptide A and/or peptide B, but three sera comprising HBV without ACA and five sera comprising HCV without ACA were found to be reactive to neither peptide. Centromere localization of CENP-A is dependent within the H3-like C-terminal website which is not autoantigenic, while the antigenic N-terminal website, which might play unidentified practical roles, should be an important region for the induction of Schisantherin B ACA. = 73). This group reacted to 37C15 and N-1. (c) ACA group II (= 7). This group reacted only to 37C15. (d) ACA group III (= 11). This group MLNR reacted to no constructs. Good epitope mapping of the N-terminus of CENP-A Once we recognized one linear autoepitope in the amino acid sequence 3C17 in the previous study [17], this sequence should symbolize an epitope in amino acids 1C19. To map the linear epitope finely in the amino acid sequence 19C52, eight ACA+ sera showing strong reactivity with the recombinant 37C15 were tested for reactivity against 20 consecutive cellulose-bound linear peptides of 10 amino acids in length and an eight amino acid overlap in the region of amino acids 11C58 of CENP-A. Seven of these eight sera showed significant reactions, mainly against the region of amino acids 25C38, as demonstrated in Fig. 3, which is a representative dot assay result. Sera of one normal donor and an anti-CENP-A?ACA in the immunoblotting analysis did not react with any of the cellulose-bound CENP-A peptides. According to the result, the amino acid 25C38 portion was considered to be a possible representative epitope in the 19C52 portion. Open in a separate windowpane Fig. 3 Reactivity of a representative anti-CENP-A+ serum with peptides spanning the N-terminal CENP-A protein (11C58 aa) by linear epitope mapping. Each spot represents Schisantherin B a linear synthetic peptide of 10-aa size bound to a cellulose membrane. The numbers 1, 2, 3, , 20, show the individual peptides, 11C20 aa, 13C22 aa, 15C24 aa, , 49C58 aa. Marked positive reactions were detected in the spot figures 8C10 (the region of 25C38 aa). Immunoreactivities of ACA+ sera to both peptides in ELISA With this study, the sequence of amino acids 25C38 named peptide B was synthesized in addition to amino acids 3C18 (peptide A) like a positive control. ELISA with each peptide conjugated to BSA was performed, and ideals 2 s.d. above the imply of 30 normal controls were identified as cut-off ideals. Twenty sera from atopic dermatitis individuals without ACA and 10 sera from SSc individuals without ACA were reactive to neither peptide A nor peptide B. Among 91 ACA+ sera, 78 sera were positive for peptide A, 79 sera for peptide B, and 73 sera were positive for both (Fig. 4). Of 13 anti-peptide A? ACA, eight were not reactive to 37C15 and the additional four were reactive to peptide B and N-1 in immunoblotting. The additional one reacted to 37C15 but did not react to N-1, which might imply the possible existence of a minor epitope around amino acid 19. None of 12 anti-peptide B? ACA was reactive to N-1, which suggests that peptide B was the most immunodominant epitope in the region of amino acids 19C52. Judging from these results, the two sequences, peptides A and B, composed the major linear Schisantherin B autoepitopes of CENP-A. Open in a separate windowpane Fig. 4 Binding assay in ELISA of peptide A and peptide B. Reactivities of anti-centromere autoantibody (ACA)-positive sera (= 91) against each peptide A and B were plotted. and axes represent reactivities to peptides A and B, respectively. The cut-off collection at 100 represents 2 s.d. above the imply binding levels of 30 normal control sera for both peptides. There was no correlation between the antibody titres against peptide A and the clinical settings. However,.