Generation of single domain antibody fragments derived from camelids and generation of manifold constructs

Methods Mol Biol. 2012:907:145-76. doi: 10.1007/978-1-61779-974-7_8.

Abstract

Immunizing a camelid (camels and llamas) with soluble, properly folded proteins raises an affinity-matured immune response in the unique camelid heavy-chain only antibodies (HCAbs). The peripheral blood lymphocytes of the immunized animal are used to clone the antigen-binding antibody fragment from the HCAbs in a phage display vector. A representative aliquot of the library of these antigen-binding fragments is used to retrieve single domain antigen-specific binders by successive rounds of panning. These single domain antibody fragments are cloned in tandem to generate manifold constructs (bivalent, biparatopic or bispecific constructs) to increase their functional affinity, to increase specificity, or to connect two independent antigen molecules.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Bacteriophages / metabolism
  • Base Sequence
  • Camelids, New World / immunology*
  • Camelus / immunology*
  • DNA, Complementary / biosynthesis
  • Electroporation
  • Epitopes / immunology
  • Escherichia coli
  • Genetic Vectors / genetics
  • Immunization
  • Immunoglobulin Heavy Chains / genetics
  • Lymphocytes / metabolism
  • Molecular Biology / methods*
  • Molecular Sequence Data
  • Polymerase Chain Reaction
  • RNA / metabolism
  • Restriction Mapping
  • Single-Domain Antibodies / genetics*

Substances

  • DNA, Complementary
  • Epitopes
  • Immunoglobulin Heavy Chains
  • Single-Domain Antibodies
  • RNA