RETRIEVAL OF HUMAN-ANTIBODIES FROM PHAGE-DISPLAY LIBRARIES USING ENZYMATIC CLEAVAGE

A combinatorial human IgG1,kappa gene library of 2 x 10(7) clones was constructed from a pericolic lymph node using the phagemid vector pCom b3H. Fabs with binding activity against tetanus toroid (TT) and keyhol e limpet hemocyanin (KLH) were isolated from this library, and one suc h TT binding Fab was used to further evaluate a new phagemid vector fo r the display of recombinant antibody fragments (MCO1). This vector wa s designed to incorporate a cleavage site for the enzyme Genenase I, a myc peptide tag, and an amber codon between the heavy chain cloning s ite and the truncated M13 phage gene III. When MCOI phage displaying a n anti-TT Fab were bound to TT on a solid substrate, elution with Gene nase I at concentrations of 5-10 mu g/ml proved as effective as acid e lution in releasing bound phage. Furthermore, enzymatic elution with G enenase I was comparable to acid elution in the enrichment of a TT bin ding Fab from the pericolic library subcloned into the vector MCO1. Im portantly, the use of enzymatic or acid elutions resulted in the retri eval of different anti-TT Fabs from this same library. We conclude tha t panning of phage-displayed combinatorial antibody libraries can be s uccessfully performed using enzymatic elution, and that this offers a useful alternative to currently available phage elution techniques.