Arthritis and rheumatism · 1996
Epitope analysis of the major reactive region of the 100-kd protein of PM-Scl autoantigen.
СтатьяПоддержка исследования: вне правительства СШАПоддержка правительства СШАПоддержка службы здравоохранения СШАНазвание и аннотация: Перевод в очереди
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Ge Q, Wu Y, James J A, Targoff I N
Аннотация
OBJECTIVE: To localize the epitope(s) bound by anti-PM-Scl antibodies in the N-terminal half of the 100-kd protein, the major antigen of the PM-Scl complex.
METHODS: Investigations were performed by immunoblotting 20 anti-PM-Scl positive sera against bacterially expressed, polymerase chain reaction-derived deletion mutants of the S1 fragment (amino acids 11-437), enzyme-linked immunosorbent assay (ELISA) screening against synthesized serial octapeptides, and ELISA screening, with anti-PM-Scl positive sera, against a synthesized 21-amino acid peptide covering the active region.
RESULTS: Anti-PM-Scl positive sera retained full immunoblot activity with fragment 207-436 and most activity with fragment 11-241, but had markedly decreased activity against fragments 236-436 and 11-212, indicating a major epitope in the aa 207-241 region. Fusion proteins with smaller fragments localized this activity between aa 226 and aa 246. Of 42 anti-S1-positive, anti-PM-Scl positive sera tested by ELISA against a synthetic peptide of this region, 36 were definitely positive, 4 borderline, and 2 negative. Similar activity was seen with a peptide from which proline 228 was deleted. Three additional epitope areas were found in S1, but each reacted with only a few sera. Anti-PMScl positive sera did not react with any octapeptide spanning the major epitope area (aa 207-246).
CONCLUSION: The main immunoblot epitope of the PM-Scl 100-kd protein is within a central area of 21 aa (aa 226-246), but is longer than the usual linear epitope. This peptide may be useful in patient testing. Three minor epitopes in S1 may also be recognized by some sera.
METHODS: Investigations were performed by immunoblotting 20 anti-PM-Scl positive sera against bacterially expressed, polymerase chain reaction-derived deletion mutants of the S1 fragment (amino acids 11-437), enzyme-linked immunosorbent assay (ELISA) screening against synthesized serial octapeptides, and ELISA screening, with anti-PM-Scl positive sera, against a synthesized 21-amino acid peptide covering the active region.
RESULTS: Anti-PM-Scl positive sera retained full immunoblot activity with fragment 207-436 and most activity with fragment 11-241, but had markedly decreased activity against fragments 236-436 and 11-212, indicating a major epitope in the aa 207-241 region. Fusion proteins with smaller fragments localized this activity between aa 226 and aa 246. Of 42 anti-S1-positive, anti-PM-Scl positive sera tested by ELISA against a synthetic peptide of this region, 36 were definitely positive, 4 borderline, and 2 negative. Similar activity was seen with a peptide from which proline 228 was deleted. Three additional epitope areas were found in S1, but each reacted with only a few sera. Anti-PMScl positive sera did not react with any octapeptide spanning the major epitope area (aa 207-246).
CONCLUSION: The main immunoblot epitope of the PM-Scl 100-kd protein is within a central area of 21 aa (aa 226-246), but is longer than the usual linear epitope. This peptide may be useful in patient testing. Three minor epitopes in S1 may also be recognized by some sera.