The combined use of high performance liquid chromatography and immuno-biochemical techniques for protein isolation: a new approach for identificationof an individual protein from a pool of proteins

HPLC was used in combination with immune-bead separation technique for iden tification of an individual protein from a pool of proteins. This was carri ed out using an in-house monoclonal antibody (ATC2) specific for placental alkaline phosphatase (PLAP) as a primary antibody for conjugation to CNBr b eads. The phosphatase activity (ALP) of FLAP was measured by colorimetric a ssay (MEDC). The data from this study has so far indicated that: 1. HPLC analysis of molecules following isolation with ATC2-conjugated bead s showed high degree of purity. This could be achieved using protein mixtur es prepared from lysates of tumour cell lines or tumour fragments. 2. HPLC-isolated FLAP maintained phosphatase activity. 3. Out of the four dissociation reagents used, diethyl amine (DEA) was foun d to be the best reagent for dissociation of antigen, ie FLAP, but not mAb from CNBr beads. 4. The profile of ALP activity was different for samples prepared from test is and kidney fragments, both in terms of the HPLC peak profile as well as the sensitivity. These data confirmed that the immune-bead separation technique in conjuncti on with HPLC were powerful tools for identifying an individual protein from a pool of proteins. These approaches are being used for the identification of FLAP molecules, as a tumour marker in patients suspected of testicular malignancies with equivocal ultrasound. Copyright (C) 2000 John Wiley & Son s, Ltd.