Selection of phage displayed antibodies based on kinetic constants.
The display of antibody fragments on the surface of filamentous bacteriophages and the selection of binders from antibody libraries have provided powerful tools to generate human antibodies. We reported recently a new concept (SAP system) for the selection of specific phages by linking antigenic recognition and phage replication, using a soluble fusion protein containing the antigen and a fragment of the M13 coat protein 3. In this investigation, a model library has been composed using six different antibody fragments which were characterized individually regarding their kass, kdiss and Ka. All Fab fragments were specific for a 15 amino acid region of the V3 loop of gp120 (HIV-1). We demonstrated that the SAP system could discriminate between the kinetic parameters of each clone, using different selection strategies. Phages expressing high affinity clones were selected preferentially using low doses of antigen but clones of lower affinity also could be selected by increasing the antigen concentration or using a preselection procedure. Phages expressing antibody fragment with high association or low dissociation rate constants were retrieved by utilizing short contact times between antigen and antibody or antigen-chase conditions.
Dueñas
M
M
Department of Immunotechnology, Lund University, Sweden.
Malmborg
A C
AC
Casalvilla
R
R
Ohlin
M
M
Borrebaeck
C A
CA
D000915:Antibody Affinity / Q000235:genetics; D017104:Bacteriophage M13 / Q000235:genetics* / Q000378:metabolism*; D001483:Base Sequence; D003001:Cloning, Molecular; D015723:Gene Library; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000235:genetics / Q000378:metabolism; D007128:Immunoglobulin Fragments / Q000235:genetics* / Q000378:metabolism*; D007700:Kinetics; D018448:Models, Immunological; D008969:Molecular Sequence Data; D011993:Recombinant Fusion Proteins / Q000378:metabolism
D007140:Immunoglobulin Fab Fragments; D007128:Immunoglobulin Fragments; D011993:Recombinant Fusion Proteins