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

HPLC was used in combination with immuno-bead separation technique for identification of an individual protein from a pool of proteins. This was carried out using an in-house monoclonal antibody (ATC2) specific for placental alkaline phosphatase (PLAP) as a primary antibody for conjugation to CNBr beads. The phosphatase activity (ALP) of PLAP was measured by colorimetric assay (MEDC). The data from this study has so far indicated that: 1. HPLC analysis of molecules following isolation with ATC2-conjugated beads showed high degree of purity. This could be achieved using protein mixtures prepared from lysates of tumour cell lines or tumour fragments. 2. HPLC-isolated PLAP maintained phosphatase activity. 3. Out of the four dissociation reagents used, diethyl amine (DEA) was found to be the best reagent for dissociation of antigen, ie PLAP, but not mAb from CNBr beads. 4. The profile of ALP activity was different for samples prepared from testis and kidney fragments, both in terms of the HPLC peak profile as well as the sensitivity. These data confirmed that the immuno-bead separation technique in conjunction with HPLC were powerful tools for identifying an individual protein from a pool of proteins. These approaches are being used for the identification of PLAP molecules, as a tumour marker in patients suspected of testicular malignancies with equivocal ultrasound.