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 pComb3H. Fabs with binding activity against tetanus toxoid (TT) and keyhole limpet hemocyanin (KLH) were isolated from this library, and one such TT binding Fab was used to further evaluate a new phagemid vector for the display of recombinant antibody fragments (MCO1). This vector was designed to incorporate a cleavage site for the enzyme Genenase I, a myc peptide tag, and an amber codon between the heavy chain cloning site and the truncated M13 phage gene III. When MCO1 phage displaying an anti-TT Fab were bound to TT on a solid substrate, elution with Genenase I at concentrations of 5-10 micrograms/ml proved as effective as acid elution in releasing bound phage. Furthermore, enzymatic elution with Genenase I was comparable to acid elution in the enrichment of a TT binding Fab from the pericolic library subcloned into the vector MCO1. Importantly, the use of enzymatic or acid elutions resulted in the retrieval of different anti-TT Fabs from this same library. We conclude that panning of phage-displayed combinatorial antibody libraries can be successfully performed using enzymatic elution, and that this offers a useful alternative to currently available phage elution techniques.
Ward
R L
RL
Department of Medical Oncology, St Vincent's Hospital, Darlinghurst, NSW, Australia.
Clark
M A
MA
Lees
J
J
Hawkins
N J
NJ
D000368:Aged; D000595:Amino Acid Sequence; D017104:Bacteriophage M13 / Q000235:genetics*; D001483:Base Sequence; D005260:Female; D015723:Gene Library; D005822:Genetic Vectors / Q000276:immunology / Q000502:physiology; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000235:genetics / Q000302:isolation & purification* / Q000502:physiology; D008198:Lymph Nodes / Q000737:chemistry; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000276:immunology / Q000302:isolation & purification*; D013381:Subtilisins
D007140:Immunoglobulin Fab Fragments; D011994:Recombinant Proteins; D013381:Subtilisins