Isolation and Epitope Mapping of Staphylococcal Enterotoxin B Single-Domain Antibodies

Abstract

Single-domain antibodies (sdAbs), derived from the heavy chain only antibodies found in camelids such as llamas have the potential to provide rugged detection reagents with high affinities, and the ability to refold after denaturation. We have isolated and characterized sdAbs specific to staphylococcal enterotoxin B (SEB) which bind to two distinct epitopes and are able to function in a sandwich immunoassay for toxin detection. Characterization of these sdAbs revealed that each exhibited nanomolar binding affinities or better. Melting temperatures for the sdAbs ranged from approximately 60 °C to over 70 °C, with each demonstrating at least partial refolding after denaturation and several were able to completely refold. A first set of sdAbs was isolated by panning the library using adsorbed antigen, all of which recognized the same epitope on SEB. Epitope mapping suggested that these sdAbs bind to a particular fragment of SEB (VKSIDQFLYFDLIYSI) containing position L45 (underlined), which is involved in binding to the major histocompatibility complex (MHC). Differences in the binding affinities of the sdAbs to SEB and a less-toxic vaccine immunogen, SEBv (L45R/Y89A/Y94A) were also consistent with binding to this epitope. A sandwich panning strategy was utilized to isolate sdAbs which bind a second epitope. This epitope differed from the initial one obtained or from that recognized by previously isolated anti-SEB sdAb A3. Using SEB-toxin spiked milk we demonstrated that these newly isolated sdAbs could be utilized in sandwich-assays with each other, A3, and with various monoclonal antibodies.

Optimizing Nanoplasmonic Biosensor Sensitivity with Orientated Single Domain Antibodies

“…The C8 sdAb, lacking the upper hinge region, was cloned into pET22b for protein expression. Protein was purified using immobilized metal affinity chromatography followed by size exclusion chromatography on an FPLC as previously described [ 26 , 30 ]. Protein sequences of all sdAb constructs are detailed in the Supplementary Material .…”

“…SdAb, which recognize a plethora of targets, have been developed, and orientated sdAb have recently been demonstrated to improve SPR biosensor sensitivity [ 23 , 24 ]. Our primary focus has been on the development of robust reagents for the detection of bio-threat agents [ 25 , 26 ]. Of those reagents, the sdAb which bind ricin have been the most studied, and thus make well defined tools for testing the utility of sdAb in novel sensor applications [ 27 , 28 ].…”

Next-Generation Sequencing of a Single Domain Antibody Repertoire Reveals Quality of Phage Display Selected Candidates

“…This study uses a library previously constructed and characterized [ 20 ]. Total RNA was isolated from the white blood cells collected from the blood of an SEB-immunized llama according to manufacturer’s protocol (Qiagen RNA Blood Mini Kit).…”

“…The following day, the culture was transferred to 500 mL of fresh broth and grown for 3 h at 30°C. Protein expression was induced with IPTG at a final concentration of 0.5 mM at 30°C for 3 h. Cells were harvested by centrifugation and the protein was harvested from the periplasm by osmotic shock and purified by immobilized metal affinity chromatography and size exclusion chromatography, as described previously [ 20 , 25 ]. Gene sequences for Aa, Ac, Ad, and Ca through Cd are available in GenBank under accession numbers KU508539 through KU508545.…”

“…It would be desirable to study similar antibodies which are present but not recovered by phage-display selection. By applying NGS to evaluate the cDNA repertoire from a previously developed sdAb library derived from a llama immunized with staphylococcal enterotoxin B (SEB) [ 20 ] and using previously identified sequences to query the entire repertoire of sequences, we sought to identify novel sdAb sequences and to characterize their stability and binding kinetics. By comparing groups of related antibodies recovered by phage display selection and by sequencing we were able to investigate the quality of the selection method.…”