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Epitope analysis of Span-1 and DUPAN-2 was compared with that of CA19-9 using the synthesized glycoconjugate sialyllacto-N-fucopentaose II (SLF11, sialyl-Lewisc) and its precursor, sialyllact-N-tetraose (LSTa, sialyl-Lewis”), conjugated to human serum albumin. The CA19-9 and DUPAN-2 assay systems specifically recognized SLF II and LSTa, respectively. The Span-1 assay system recognized both SLF IIIand LSTa, although the reactivity with the former was far stronger than that with the latter. These results were, in general, compatible with those obtained from assaying these markers in the sera of two pancreatic cancer patients with definite Lewis-negative phenotype and in the sera of 39 CA19-9-negative pancreatic cancer patients. In conclusion, DUPAN-2 is the precursor of CA19-9 and is accumulated in the sera of pancreatic cancer patients with Lewis-negative phenotype and Span-1 has an advantage over CA19-9 in the diagnosis of patients with Lewis-negative phenotype, although both markers have almost the same sensitivity for this malignancy.