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

Abstract

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 Ilama. The highest-affinity sdAb isolated (CSF2A) had a K(D) of 5-20 microM for two IAA-protein conjugates and a K(D) of 20 microM 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 microM) 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.

VHH antibodies: emerging reagents for the analysis of environmental chemicals

“…Several proteins have been found to bind auxin amongst which is the relatively poorly studied Auxin Binding Protein (ABP). Authors hypothesized “that antibodies that bind to IAA may be used as surrogate ABPs to study the structure-activity relationships of IAA and the auxinic herbicides binding to their putative receptor.” In this work [17], authors isolated five different sdAbs from a naïve llama library with relatively low affinity for the IAA-BSA conjugate (K d ≥ 5 µM) as determined by SPR (surface plasmon resonance). Inhibition experiments with sdAb CSF2A (K d = 5 µM) revealed broad cross-reactivity with all auxinic herbicides with IC 50 values > 200 µM for herbicides and 800 µM for IAA.…”

“…Another example comes from the same group, where five selected anti-auxin VHH clones were shuffled by staggered extension process (StEP). Although none of the shuffled clones had better affinity toward auxin than the best original clone, the affinity toward auxinic herbicides was significantly decreased [17]. Recently an interesting study investigating the structure – binding relationship of an anti-methotrexate (anti-MTX) VHH antibody was published by Fanning and Horn [49, 23].…”

“…SPR techniques were used in the study by Doyle and colleagues [25] to evaluate the binding kinetics of a VHH to the mycotoxin 15-acetyldeoxynivalenol (15-AcDON) in both a monomeric and pentameric form. VHHs against methotrexate [22], picloram [16], and auxin [17] were also evaluated by SPR technology. SPR was used to evaluate binding of VHHs against TCC towards both the coating antigen, as well as the free analyte [29].…”

A new human catalytic antibody Se‐scFv‐2D8 and its selenium‐containing single domains with high GPX activity

“…Many investigations for single-domain antibodies indicated that despite the heavy chain variable fragments (V H ) lack of variable light and heavy chain pairing, they also can recognize and bind a wide variety of epitopes and antigen types (Nguyen et al, 2000;van der Linden et al, 2000;Sheedy et al, 2006;Doyle et al, 2008). Moreover, some research on the catalytic antibody also indicated that the heavy chain was found to be the determining factor for catalytic efficiency (Ulrich and Schultz, 1998).…”

Multiplexed Fluid Array Screening of Phage Displayed Anti-Ricin Single Domain Antibodies for Rapid Assessment of Specificity

“…Others have focused on creating naive phage display libraries from which it is theoretically possible to extract binders for a nearly boundless number of targets (15,16); however, naive libraries may possess only low-affinity binders. While most applications require high-affinity binders, lower affinity binders may provide the starting point for affinity maturation.…”