Nonradioactive immunoassays incorporating an element of amplification in their detection system require the use of components that are highly purified. Flavin adenine dinucleotide-3'-phosphate (FADP) is the primary substrate used in such an amplification assay. For incorporation into a simple, single-pot assay system, the concentration of contaminating flavin adenine dinucleotide (a prosthetic group for the enzyme D-aminoacid oxidase used in the amplification cascade assay) in this primary substrate must be minimized to achieve maximum sensitivity. Production of the substrate to a high degree of purity has been achieved using apo-glucose oxidase to specifically remove contaminating flavin adenine dinucleotide from solution and hydrolysis of a cyclic intermediate as a final production protocol by ribonuclease T2 to give the product in high yield. The use of continuous ultrafiltration reactors at each stage is described and compared to a final production step utilizing immobilized ribonuclease T2. These reactors allow large volumes of material to be handled and assist in the scale-up of these processes. The suitability of each protocol is assessed for the commercial production of FADP.