title: Hierarchical affinity maturation of a phage library derived antibody for the selective removal of cytomegalovirus from plasma.
abstract: Recombinant antibody fragments can be produced in large quantities using bacterial expression systems and could potentially be useful for the generation of biofilters for the selective removal of viral particles from fluids. A human single chain-Fv antibody library, derived from synthetic repertoires of germ line VH-gene segments rearranged in vitro and paired to a single light chain (Nissim et al., 1994, EMBO J., 13, 692-698), has recently been used to isolate hundreds of different binding specificities by panning with antigen. Antibodies from this library typically have affinities in the 10(6)-10(7) M-1 range. Occasionally, better binders are isolated but at other times the affinities recovered are poor. In the latter situation binding cannot be detected with soluble antibodies, but only by high-avidity display of multiple copies of antibodies on phage. By panning with human cytomegalovirus (HCMV)-coated immunotubes, we have isolated a number of antibody clones from this library that bound to the antigen only if displayed on the filamentous phage, but not in soluble form. One of these clones was selected for an affinity maturation procedure, achieved by combinatorial mutagenesis of the complementarity determining region 3 (CDR3) of the antibody light chain, followed by selection of the resulting library for HCMV binding. By this means, we were able to isolate a number of binders, some of which exhibited specific HCMV binding in soluble form. The clone that gave the strongest ELISA signal was expressed in bacteria, purified in solution, characterised using a novel capture methodology with surface plasmon resonance detection on a BIAcore instrument and used for the production of an immunofilter for the removal of HCMV form human serum. The filter removed more than 99% of applied HCMV in 10 min circulation time, while the amount of HCMV retained non-specifically in a cartridge derivatised with a non-specific antibody was less than 10% under similar conditions.
authors.affiliation: Dipartimento di Biologia Molecolare, Università di Siena, Centro Didattico del Policlinico Le Scotte, Italy. nerip@unisi.it
authors.forename: A
authors.initials: A
authors.lastname: Pini
authors.forename: A
authors.initials: A
authors.lastname: Spreafico
authors.forename: R
authors.initials: R
authors.lastname: Botti
authors.forename: D
authors.initials: D
authors.lastname: Neri
authors.forename: P
authors.initials: P
authors.lastname: Neri
mesh_terms: D000914:Antibodies, Viral / Q000235:genetics / Q000302:isolation & purification*; D000915:Antibody Affinity / Q000235:genetics; D001483:Base Sequence; D001709:Biotechnology; D003001:Cloning, Molecular; D003587:Cytomegalovirus / Q000276:immunology* / Q000302:isolation & purification; D005374:Filtration; D006801:Humans; D007128:Immunoglobulin Fragments / Q000235:genetics; D007135:Immunoglobulin Variable Region / Q000235:genetics; D017912:Inovirus / Q000235:genetics / Q000276:immunology*; D008969:Molecular Sequence Data; D010949:Plasma / Q000821:virology*; D011994:Recombinant Proteins / Q000276:immunology / Q000302:isolation & purification
chemical_list: D000914:Antibodies, Viral; D007128:Immunoglobulin Fragments; D007135:Immunoglobulin Variable Region; D011994:Recombinant Proteins