Two mouse monoclonal antibodies (MoAbs), KM-93 raised against human lung adenocarcinoma and KM-231 raised against human gastric cancer, were useful in serum diagnosis of several human cancer. KM-93 and KM-231 recognize sialyl Lex epitope and sialyl Lea epitope, respectively, expressed on glycoprotein and glycolipid. We established a new "cocktail" sandwich enzyme-linked immunosorbent assay system using the two MoAbs and the advantage of this assay system, which can simultaneously detect sialyl Lex and sialyl Lea antigens, is assessed in the present study. The new assay system is composed of a mixture of KM-93 and KM-231 as 1st antibodies and a mixture of biotinylated two MoAbs as 2nd antibodies. We evaluated the concentration of MoAbs and optimized it to gain high cancer-positivity. This assay system covered sialyl Lex positive and/or sialyl Lea-positive sera and gave a high rate of positive results in lung adenocarcinoma (62.3%), gastric cancer (32.5%), colon cancer (37.5%), pancreatic cancer (83.3%), bile duct and gall bladder cancer (66.7%) and hepatoma (76.9%), whereas positive results in healthy adults remained low. Positive results in benign diseases of lung (12.5%), pancreas (10.8%), gall bladder and bile duct (9.1%) were very low, but were higher in liver cirrhosis (33.3%), hepatitis and liver injury (34.8%). Simultaneous detection of two carbohydrate antigens, sialyl Lex and sialyl Lea was clearly superior to single detection.