The 2B3 A2 cys-diabody, (cDb, 50 kDa) was developed and validated for preclinical in vivo targeting of PSCA at UCLA (30 (link)). It was derived by yeast affinity maturation of a humanized monoclonal anti-PSCA antibody, 2B3, and engineered to contain a C-terminal free cysteine that forms an inter-chain disulfide bond stabilizing dimerization. Upon mild reduction this disulfide bond can be broken and free thiols are available for site-specific labeling away from the antigen binding site using e.g. maleimide chemistry. A2 cDb was purified from mammalian cell culture supernatant using immobilized metal affinity chromatography. Protein concentrations were determined photometrically and purity was analyzed by SDS-PAGE. Detailed biodistribution data for the A2 cDb was previously determined (21 ). Non-specific binding was not seen. Fluorescent signals were present in liver, kidney and bladder due to the metabolism and urinary excretion of the probe. Cy5 Maleimide (649 nm absorbance, 670 nm emission) was purchased from GE Healthcare (Piscataway, NJ).