Application of a recombinant Fab fragment from a phage display library forsensitive detection of a target antigen by an inhibition ELISA system

We have found that the recombinant Fab (rFab) produced by phage display sys tem was detectable for a target antigen more sensitive than the parental mo noclonal antibody (MoAb). The Fab phage display library was constructed fro m hybridoma cells producing APU-6 MoAb specific for a modified nucleoside, pseudouridine that have been studied as a urinary marker for malignancy. Fa b-displayed phage clones were screened by a direct ELISA, and the single po sitive clone was finally obtained. Although the reaction pattern of rFab ag ainst pseudouridine and uridine was almost identical to that of MoAb, detec tion sensitivity of rFab was approximately 30 times higher than that of MoA b. Since the sensitivity of rFab was almost identical to that of Fab fragme nt prepared by papain digestion of MoAb, the increased sensitivity is consi dered to be the nature of Fab fragment. The sensitivity of established assa y system was sufficient for quantitative determination of serum pseudouridi ne levels in healthy individuals and cancer patients. This procedure may be applicable for improvement of detection sensitivity of a MoAb-based inhibi tion ELISA system for drugs or low molecular weight compounds. (C) 1999 Els evier Science B.V. All rights reserved.