Immunoaffinity isolation of CEACAM1 on hydrazide-derivatized cellulose with immobilized monoclonal anti-CEA antibody

Carcinoembryonic cell adhesion molecule 1 (CEACAM1) is a human membrane gly coprotein belonging to the carcinoembryonic antigen (CEA) family and to the immunoglobulin superfamily. It is expressed in apical membranes of many ep ithelial cells in gastrointestinal and urogenital tract and also in granulo cytes and lymphocytes, and its biological effect in human tissues has recen tly been discussed in literature. The purpose of this study was to isolate CEACAM1 glycoprotein from bile and characterize its purity and recovery whi ch has not been described before. Affinity chromatography of CEACAM1 on hyd razide-activated cellulose with immobilized monoclonal anti-CEA F34-187 ant ibody is described. The immunoglobulin carbohydrate moiety was oxidized by periodate and then bound to hydrazide-activated matrix. Crude protein fract ion from bile was applied on the affinity column and after extensive washin g of non-bound proteins CEACAM1 was eluted with 6 M guanidine-HCl. A single immunopositive 85 kDa band was detected on Western blots with anti-CEA ant ibody after SDS-PAGE. We found out that CEACAM1 was not stainable with any common method of protein staining and the only non-specific method which co uld detect the 85 kDa band was a lectin staining. Copyright (C) 2001 John W iley & Sons, Ltd.