Auxin-specific single-domain antibodies: Isolation, engineering and characterization

Indole-3-acetic acid-specific single-domain antibodies were isolated from a naive llama phage-display library. After three rounds of panning, five single-domain antibodies were selected. The five single-domain antibodies isolated from the naive phage-display library were shuffled by staggered extension process (StEP) to determine which complementary-determining regions (CDRs) were instrumental in binding the hapten. Indole-3-acetic acid-specific single-domain antibodies were also isolated from a llama naive ribosome-display library. After five rounds of panning, three single-domain antibodies were selected for further characterization.; The single-domain antibodies isolated from the phage-display, shuffled and ribosome-display libraries were fused to the Escherichia coli B subunit to form pentabodies. Single-domain antibodies and pentabodies were characterized by surface plasmon resonance. The single-domain antibodies isolated from the phage-display library were analyzed for binding to an indole-3-acetic acid-BSA conjugate, as well as for binding free indole-3-acetic acid and several synthetic auxin analogues (1-naphtalene acetic acid and the following auxinic herbicides; clopyralid, dicamba, 2,4-dichlorophenoxyacetic acid, MCPA, mecoprop and quinclorac) through competitive inhibition experiments. The highest-affinity binder has a KD for free indole-3-acetic acid of 6 muM, and cross-reacted with all auxinic herbicides tested.; Shuffling of the five original single-domain antibodies isolated from the phage-display library resulted in novel antibodies with CDRs originating from either one, two or three parental clones. Clones with point mutations were also obtained. The shuffled clones isolated showed a marked increase in specificity but not in affinity for free indole-3-acetic acid. Differential affinity of the mutant clones for indole-3-acetic acid showed that CDR2 is the most likely CDR imparting binding to the hapten.; These results demonstrate that hapten-binding single-domain antibodies can be isolated from phage- and ribosome-display libraries. Pentamerization of these single-domain antibodies allows for better characterization by surface plasmon resonance. Furthermore, mutagenesis by staggered extension process is a suitable strategy to identify structure-function relationships.