Abstract
ABTAG is a camelid single-domain antibody (sdAb) that binds to bovine serum albumin (BSA) with low picomolar affinity. In surface plasmon resonance (SPR) analyses using BSA surfaces, bound ABTAG can be completely dissociated from the BSA surfaces at low pH, over multiple cycles, without any reduction in the capacity of the BSA surfaces to bind ABTAG. A moderate throughput, SPR-based, antibody screening assay exploiting the unique features of ABTAG is described. Anti-carcinoembryonic antigen-related cell adhesion molecule 6 (CEACAM6) sdAbs were isolated from a phage-displayed sdAb library derived from the heavy chain antibody repertoire of a llama immunized with CEACAM6. Following one or two rounds of panning, enriched clones were expressed as ABTAG fusions in microtiter plate cultures. The sdAb-ABTAG fusions from culture supernatants were captured on BSA surfaces and CEACAM6 antigen was then bound to the captured molecules. The SPR screening method gives a read-out of relative expression levels of the fusion proteins and kinetic and affinity constants for CEACAM6 binding by the captured molecules. The library was also panned and screened by conventional methods and positive clones were subcloned and expressed for SPR analysis. Compared to conventional panning and screening, the SPR-based ABTAG method yielded a considerably higher diversity of binders, some with affinities that were three orders of magnitude higher affinity than those identified by conventional panning.
Highlights
Over the past 25 years, in vitro display technologies, most notably phage and yeast display, have increasingly become the methods of choice for the isolation and affinity maturation of monoclonal antibody fragments
Immunization of a llama with recombinant carcinoembryonic antigen-related cell adhesion molecule 6 (CEACAM6) resulted in strong immune response against the immunogen as indicated by ELISA comparing the preimmune and postimmunization sera collected on day 71 from the animal (Figure S1 in Supplementary Material)
A phage-displayed VHH library with a functional size of 2.9 × 107 clones was constructed from the lymphocytes of the immunized llama and used to select CEACAM6-binding single-domain antibody (sdAb)
Summary
Over the past 25 years, in vitro display technologies, most notably phage and yeast display, have increasingly become the methods of choice for the isolation and affinity maturation of monoclonal antibody fragments. The classical protocol for isolating antibody fragments against a given target by phage display technology generally includes immunizing an animal, measuring the immune response, constructing a phage-displayed antibody fragment library and performing three to five rounds of panning to enrich for clones that bind to the target. This generally gives a manageable number of clones for subsequent expression, purification and characterization. This is a relatively slow, costly and laborious process that generally yields no more than a dozen binders, and often fewer
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