Development of Antiricin Single Domain Antibodies Toward Detection and Therapeutic Reagents

Abstract

Single domain antibodies (sdAb) that bind ricin with high affinity and specificity were selected from a phage display library derived from the mRNA of heavy chain antibodies obtained from lymphocytes of immunized llamas. The sdAb were found to recognize three distinct epitopes on ricin. Representative sdAb were demonstrated to function as both capture and tracer elements in fluid array immunoassays, a limit of detection of 1.6 ng/mL was obtained. One sdAb pair in particular was found to be highly specific for ricin. While polyclonal antibodies cross react strongly with RCA120, the sdAb pair had minimal cross reactivity. In addition, the binders were found to be thermal stable, regaining their ricin binding activity following heating to 85 degrees C for an hour. Cycles of thermally induced unfolding of the sdAb and their subsequent refolding upon cooling was monitored by circular dichroism. As several of the sdAb were observed to bind to ricin's A chain, cell free translation assays were performed to monitor the ability of the sdAbs to inhibit ricin's biological activity. One of the sdAb (C8) was particularly effective and blocked ricin's biological activity with an effectiveness equal to that of a mouse antiricin antibody. These results indicate that antiricin sdAb have great potential for both diagnostic and therapeutic applications.

Integrating scFv into xMAP Assays for the Detection of Marine Toxins

“…The Luminex xMAP methodology consists of assays performed on color coded beads that can be highly multiplexed. Both sandwich and competitive format immunoassays have been demonstrated using the Luminex system [17,45,46]. In this article, an xMAP assay monitored using a MAGPIX system for the individual detection of STX and DA utilizing scFv recognition elements was demonstrated, Figure 4, Figure 5, and Figure S1.…”

Polymer-antibody fragment conjugates for biomedical applications

“…Halogen-containing polymers can be used to couple with sulfhydrylcontaining antibody fragments via displacement of the halogen group by an attacking nucleophile (sulfhydryl from the protein), forming an alkylated derivative with the loss of HX (where X is the halogen and the hydrogen comes from the nucleophile) (19). This reaction lacks specificity, however, and can also forma stable compound with ε-amines of lysines or the imidazole ring or thioether group of methionine residues.Iodoacetate exhibits greater specificity forsulfhydryl cysteine residues, with maleimide groupsthe most popular constituent of many heterobifunctional cross-linking agents used to form thioether bonds with cysteine-containing proteins.The unsaturated double bond of maleimide-containing polymers can undergo an alkylation reaction with sulfhydryl groups to form stable thioether bondswith proteins (20). The reactionis highly specific for thiol groups at pH 6.5-7.5.…”

“…Isolated antibodies can be readilyaffinity maturedto improve their binding and engineered to create smaller, antibody-derived fragments of <10 nm dimensions that can be further modified to facilitate extensive chemical functionalization. This has led to increasing encroachment of fragments into lucrative fields traditionally associated with monoclonal antibody-polymer conjugates, such as therapeutics where humanized and PEGylatedantibody fragments have gained regulatory approval in recent yearsfor treatment of Crohn's disease and rheumatoid arthritis ( [15]; reviewed in [16]), and immunodetection, where small, easily functionalized fragments are fast becoming the molecules of choice for in vitro detection and monitoring applications [17][18][19][20] and, somewhat more cautiously, in vivoimaging and diagnostics [21,22].…”

Selection and evaluation of single domain antibodies toward MS2 phage and coat protein

“…Direct binding and sandwich assays were performed essentially as described previously (Anderson et al, 2008;Liu et al, 2007). To conduct direct binding assay, CP, phage and the non-related antigen, Bacillus anthracis Large Antigen (BCLA), were immobilized on MagPlex carboxylated microspheres (Luminex, Austin, TX) using the two-step carbodiimide coupling protocol provided by the manufacturer.…”