Selection, Characterization, and CDR Shuffling of Naive Llama Single-Domain Antibodies Selected against Auxin and Their Cross-Reactivity with Auxinic Herbicides from Four Chemical Families

Indoleacetic acid (IAA)-binding single-domain antibodies (sdAbs) were isolated from a naive phage-display library constructed from the heavy chain antibody repertoire of a llama. The highest-affinity sdAb isolated (CSF2A) had a K_D of 5−20 μM for two IAA−protein conjugates and a K_D of 20 μM for free IAA. This sdAb also bound to a synthetic auxin analogue, 1-naphthaleneacetic acid (NAA), and to six auxinic herbicides (K_D values of 0.5−2 mM), but not to serotonin and tryptophan, which are structurally similar to IAA but have no auxinic activity. To understand how sdAb CSF2A binds IAA and to determine which complementary-determining region(s) (CDR) participate(s) most in binding IAA, CSF2A was shuffled with four other sdAb clones by staggered extension process (StEP). After panning against IAA, two shuffled sdAbs were found:  sdAb CSB1A, which originated from three different parental clones, and sdAb CSE8A, derived from two parental clones. These shuffled sdAbs and CSF2A were each fused to the B subunit of the Escherichia coli verotoxin, resulting in the formation of the pentamerized sdAbs V2NCSB1A, V2NCSE8A, and V2NCSF2A, which were analyzed by surface plasmon resonance (SPR) along with the sdAbs previously isolated. The shuffled clones had affinity for IAA (20 μM) similar to that of the highest affinity parental clone CSF2A, but much lower affinity for the auxinic herbicides. CDR2 was instrumental in binding IAA, whereas hydrophobic CDR3 was important for binding the auxinic herbicides. A novel SPR methodology is also described for specific immobilization of pentamerized sdAbs, allowing determination of K_D values of Ab interaction with underivatized, low molecular weight haptens. Keywords: Hapten; indole-3-acetic acid; llama single-domain antibodies; pentabody; surface plasmon resonance