The rescue by phage display of human Fabs to gp120 HIV-1 glycoprotein using EBV transformed lymphocytes.
Human hybridomas secreting monoclonal antibodies in a stable manner are difficult to develop. The main difficulties are the restricted techniques for B-cell immortalization, the low number of sensitized B cells in peripheral blood, and the impossibility, for ethical reasons, to immunize humans with most antigens. Phage display has proved to be a powerful method for the generation of recombinant antibody fragments. This technology relies on the construction of recombinant Fab or scFv libraries and their display on phage M13. In order to rescue unstable B-cell clones secreting human antibodies we set up a method for the selection by phage display of human IgG fragments from Epstein-Barr virus (EBV)-transformed clones and applied it to the selection by phage display of Fabs directed against HIV-1 gp120, using a seropositive blood sample. The approach combines B-cell transformation by EBV of peripheral blood lymphocytes from a seropositive donor, preselection of specific IgG anti-gp120 producing clones, and the construction of a targeted human antibody library. In this library the percentage of heavy and light chain coding sequences expressed in Escherichia coli, amplified by a set of specific 5' primers for different antibody germ lines, was similar to that observed with the original untransformed B-cell sample. One round of panning was sufficient for the rescue of three Fabs specific for HIV-1 gp120 protein, which proves the efficiency of this technique.
Kempf
E
E
Laboratoire d'immunotechnologie et microbiologie moléculaires, UPRES 1329, Ecole Supérieure de Biotechnologie de Strasbourg, Boulevard Sébastien Brant, 67400 Illkirch, France.
Weiss
E
E
Klein
P
P
Glacet
A
A
Spratt
S
S
Bourel
D
D
Orfanoudakis
G
G
D000595:Amino Acid Sequence; D000918:Antibody Specificity; D001402:B-Lymphocytes / Q000276:immunology / Q000821:virology; D001483:Base Sequence; D002472:Cell Transformation, Viral; D017931:DNA Primers / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D015483:HIV Antibodies / Q000096:biosynthesis / Q000235:genetics*; D015699:HIV Envelope Protein gp120 / Q000276:immunology*; D015497:HIV-1 / Q000276:immunology*; D004854:Herpesvirus 4, Human; D006801:Humans; D006825:Hybridomas / Q000276:immunology; D007140:Immunoglobulin Fab Fragments / Q000096:biosynthesis / Q000235:genetics*; D007074:Immunoglobulin G / Q000096:biosynthesis / Q000235:genetics; D066298:In Vitro Techniques; D008969:Molecular Sequence Data; D019151:Peptide Library
D017931:DNA Primers; D015483:HIV Antibodies; D015699:HIV Envelope Protein gp120; D007140:Immunoglobulin Fab Fragments; D007074:Immunoglobulin G; D019151:Peptide Library