title: Isolation of a highly thermal stable lama single domain antibody specific for Staphylococcus aureus enterotoxin B.
abstract: BACKGROUND
  Camelids 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.
  
  
  RESULTS
  Here 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.
  
  
  CONCLUSIONS
  The 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.
authors.affiliation: MITRE Corporation, McLean, Virginia 22102, USA.
authors.forename: Russell R
authors.initials: RR
authors.lastname: Graef
authors.forename: George P
authors.initials: GP
authors.lastname: Anderson
authors.forename: Katherine A
authors.initials: KA
authors.lastname: Doyle
authors.forename: Dan
authors.initials: D
authors.lastname: Zabetakis
authors.forename: Felicia N
authors.initials: FN
authors.lastname: Sutton
authors.forename: Jinny L
authors.initials: JL
authors.lastname: Liu
authors.forename: Joseline
authors.initials: J
authors.lastname: Serrano-González
authors.forename: Ellen R
authors.initials: ER
authors.lastname: Goldman
authors.forename: Lynn A
authors.initials: LA
authors.lastname: Cooper
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry / Q000276:immunology* / Q000302:isolation & purification; D000918:Antibody Specificity; D002161:Camelids, New World / Q000276:immunology*; D002942:Circular Dichroism; D004768:Enterotoxins / Q000737:chemistry / Q000276:immunology*; D005453:Fluorescence; D006358:Hot Temperature; D007118:Immunoassay; D007143:Immunoglobulin Heavy Chains / Q000737:chemistry / Q000276:immunology* / Q000302:isolation & purification; D057230:Limit of Detection; D008969:Molecular Sequence Data; D019151:Peptide Library; D058849:Protein Refolding; D017434:Protein Structure, Tertiary; D013208:Staphylococcal Toxoid / Q000276:immunology; D013211:Staphylococcus aureus / Q000737:chemistry; D044366:Transition Temperature
chemical_list: D000911:Antibodies, Monoclonal; D004768:Enterotoxins; D007143:Immunoglobulin Heavy Chains; D019151:Peptide Library; D013208:Staphylococcal Toxoid; C031818:enterotoxin B, staphylococcal