Construction and Validation of PSMA-DMAb

To construct the PSMA-DMAb , the genes of both the variable heavy (V_H) and variable light (V_L) fragments of a human anti-PSMA mAb were examined, optimized, and constructed through the use of synthetic oligonucleotides with several modifications to improve expression as previously described [15 (link)]. DNA was formulated in water for subsequent administration into mice. An empty pVax1 expression vector was used as a negative control. Cells (293T) were transfected with the PSMA-DMAb plasmid and confirmation of PSMA-DMAb binding to recombinant human PSMA was carried out by Western blot analysis. Briefly, recombinant PSMA protein (R&D systems) was run on an SDS-PAGE gel and transferred to Immobilon-PVDF membrane (EMD Millipore). Membranes were blocked for 1 h in blocking buffer (Li-Cor Biosciences) and then incubated for 1 h with either commercial anti-PSMA mAb (R&D systems), pooled day 14 sera from PSMA-DMAb plasmid-injected mice, or supernatants from PSMA-DMAb plasmid-transfected 293T cells. Membranes were washed and then incubated for 1 h with a goat anti-human IgG 680RD antibody (Li-Cor Biosciences) and washed. Protein bands were visualized by scanning membranes with a Li-Cor Odyssey CLx scanner [19 (link)].