title: Single domain antibody-alkaline phosphatase fusion proteins for antigen detection--analysis of affinity and thermal stability of single domain antibody.
abstract: Single domain antibody (sdAb)-alkaline phosphatase (AP) fusion proteins have been demonstrated to be useful immunodiagnostic reagents for bio-threat agent detection. The bivalent nature of sdAb-AP fusion proteins significantly increases effective affinity and thus the sensitivity of detection, but the thermal stability of the fusion protein had not been explored. This property is critical for the development of immunoassays for use in austere environments. In this study four sdAbs with specificity for MS2 phage coat protein (CP) were expressed as fusions with AP in order to evaluate the thermal stability and affinity of the resulting constructs. The melting temperature (Tm) of the sdAb and sdAb-AP fusion proteins was measured by a combination of Circular Dichroism (CD), differential scanning calorimetry (DSC) and Fluorescence-based Thermal Shift assay. Binding kinetics were assessed using surface plasmon resonance (SPR). Our results indicated that the AP fusion protein did not increase the Tm or enhance thermal stability of the sdAb, but did provide the expected increase in binding affinity as compared to the original sdAb.
authors.affiliation: Naval Research Laboratory, Center for Bio/Molecular Science and Engineering, 4555 Overlook Ave SW, Washington, DC 20375, USA. jinny.liu@nrl.navy.mil
authors.forename: Jinny L
authors.initials: JL
authors.lastname: Liu
authors.forename: Dan
authors.initials: D
authors.lastname: Zabetakis
authors.forename: Audrey Brozozog
authors.initials: AB
authors.lastname: Lee
authors.forename: Ellen R
authors.initials: ER
authors.lastname: Goldman
authors.forename: George P
authors.initials: GP
authors.lastname: Anderson
mesh_terms: D000469:Alkaline Phosphatase / Q000737:chemistry / Q000276:immunology* / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D000915:Antibody Affinity / Q000276:immunology*; D000941:Antigens / Q000276:immunology*; D002152:Calorimetry, Differential Scanning / Q000379:methods; D002161:Camelids, New World; D002942:Circular Dichroism / Q000379:methods; D007118:Immunoassay / Q000379:methods*; D007700:Kinetics; D008969:Molecular Sequence Data; D011993:Recombinant Fusion Proteins / Q000276:immunology*; D016415:Sequence Alignment; D061905:Single-Domain Antibodies / Q000737:chemistry / Q000276:immunology*; D020349:Surface Plasmon Resonance / Q000379:methods; D044366:Transition Temperature
chemical_list: D000941:Antigens; D011993:Recombinant Fusion Proteins; D061905:Single-Domain Antibodies; D000469:Alkaline Phosphatase