Production and Characterization of Monoclonal and Polyclonal Antibodies Against Truncated Recombinant Dickkopf-1 as a Candidate Biomarker

Abstract

Several studies have reported an increased serum level of Dickkopf (DKK-1) protein in a variety of cancers, including multiple myeloma, lung, colorectal, bone loss, and Alzheimer's disease. This protein has potential to be used as a biomarker for the diagnosis of some cancers, especially bone loss in multiple myeloma. In the present study, to measure the concentration level of DKK-1 protein, rabbit polyclonal antibody (pAb) and mouse monoclonal antibodies (mAbs) were produced against this protein. New Zealand white rabbits and BALB/c mice were immunized with the chimeric recombinant DKK-1 antigen. Immunized mouse spleen cells were fused with SP2/0 cells to generate anti-rDKK-1 antibody-producing hybridoma cells. Antibodies were purified by protein A affinity chromatography and assessed using sodium dodecyl sulfate polyacrylamide gel, western blotting and enzyme-linked immunosorbent assay. These results implied that the pAb and mAb were produced against the DKK-1 protein. The Kd value of 5 × 10 -9 M was recorded for the mAb MR6F3 toward native DKK-1, and the Ig isotype was identified as IgG2b. No cross-reactivity was shown with DKK-2 by MR6F3. Collectively, our results revealed that the produced pAb and mAb could be used in the measurement of DKK-1 protein.