Isolation of a Highly Thermal Stable Lama Single Domain Antibody Specific for Staphylococcus aureusEnterotoxin B

Abstract

BackgroundCamelids and sharks possess a unique subclass of antibodies comprised of only heavy chains. The antigen binding fragments of these unique antibodies can be cloned and expressed as single domain antibodies (sdAbs). The ability of these small antigen-binding molecules to refold after heating to achieve their original structure, as well as their diminutive size, makes them attractive candidates for diagnostic assays.ResultsHere we describe the isolation of an sdAb against Staphyloccocus aureus enterotoxin B (SEB). The clone, A3, was found to have high affinity (Kd = 75 pM) and good specificity for SEB, showing no cross reactivity to related molecules such as Staphylococcal enterotoxin A (SEA), Staphylococcal enterotoxin D (SED), and Shiga toxin. Most remarkably, this anti-SEB sdAb had an extremely high Tm of 85°C and an ability to refold after heating to 95°C. The sharp Tm determined by circular dichroism, was found to contrast with the gradual decrease observed in intrinsic fluorescence. We demonstrated the utility of this sdAb as a capture and detector molecule in Luminex based assays providing limits of detection (LODs) of at least 64 pg/mL.ConclusionThe anti-SEB sdAb A3 was found to have a high affinity and an extraordinarily high Tm and could still refold to recover activity after heat denaturation. This combination of heat resilience and strong, specific binding make this sdAb a good candidate for use in antibody-based toxin detection technologies.

Antiviral Activity of a Single-Domain Antibody Immunotoxin Binding to Glycoprotein D of Herpes Simplex Virus 2

“…To further validate the VHH sequences, they were recloned, expressed, and purified from E. coli using an osmotic shock procedure described by others (25). The purified VHH proteins were separated by SDS-PAGE for Coomassie staining to determine size and purity (Fig.…”

“…The VHH constructs were transformed into E. coli BL21(DE3) competent cells (New England BioLabs, Ipswich, MA). An osmotic shock protocol was utilized to purify the soluble VHH from the periplasmic space, as described by Graef et al (25). Briefly, an ON 30-ml midscale culture was diluted in 450 ml Terrific broth and grown at 25°C for 3 h. Cells were induced at 1 mM isopropyl ␤-D-1-thiogalactopyranoside (IPTG) (Lab Scientific, Inc., Highlands, NJ) and grown for an additional 3 h at 25°C.…”

Staphylococcal Enterotoxins, Stayphylococcal Enterotoxin B and Bioterrorism

“…Investigations using immune lymphocytes to prepare Mabs may provide therapeutics without the expense of humanization. Additionally, developing small-domain antibody fragments such as camilid antibodies may also provide therapeutic agents that, because of their size, may not be antigenic in humans (Graef et al, 2011). Further studies are needed to identify antibody reagents that will be successful therapeutics, and whether the antibody reagents will need to be combined with pharmacologic agents to enhance their efficacy.…”

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

“…Their small size, ease of expression in Escherichia coli , thermal stability, and ability to refold after denaturation have made them attractive alternatives to conventional antibodies [17]. We previously isolated an sdAb specific for SEB, however to enable sdAb-based sandwich immunoassays for SEB additional binders are required that recognized a different epitope [18]. In this work, we describe the isolation and characterization of additional sdAbs and the development of assays that utilize the newly isolated sdAbs, our previously characterized sdAb A3, and mAbs for the sensitive detection of SEB in complex matrices.…”