title: A broad set of different llama antibodies specific for a 16 kDa heat shock protein of Mycobacterium tuberculosis.
abstract: BACKGROUND
  Recombinant antibodies are powerful tools in engineering of novel diagnostics. Due to the small size and stable nature of llama antibody domains selected antibodies can serve as a detection reagent in multiplexed and sensitive assays for M. tuberculosis.
  
  
  RESULTS
  Antibodies for Mycobacterium tuberculosis (M. tb) recognition were raised in Alpaca, and, by phage display, recombinant variable domains of heavy-chain antibodies (VHH) binding to M. tuberculosis antigens were isolated. Two phage display selection strategies were followed: one direct selection using semi-purified protein antigen, and a depletion strategy with lysates, aiming to avoid cross-reaction to other mycobacteria. Both panning methods selected a set of binders with widely differing complementarity determining regions. Selected recombinant VHHs were produced in E. coli and shown to bind immobilized lysate in direct Enzymelinked Immunosorbent Assay (ELISA) tests and soluble antigen by surface plasmon resonance (SPR) analysis. All tested VHHs were specific for tuberculosis-causing mycobacteria (M. tuberculosis, M. bovis) and exclusively recognized an immunodominant 16 kDa heat shock protein (hsp). The highest affinity VHH had a dissociation constant (KD) of 4 × 10(-10) M.
  
  
  CONCLUSIONS
  A broad set of different llama antibodies specific for 16 kDa heat shock protein of M. tuberculosis is available. This protein is highly stable and abundant in M. tuberculosis. The VHH that detect this protein are applied in a robust SPR sensor for identification of tuberculosis-causing mycobacteria.
authors: Plant Research International, Wageningen, The Netherlands.
authors: Anke K
authors: AK
authors: Trilling
authors: Hans
authors: H
authors: de Ronde
authors: Linda
authors: L
authors: Noteboom
authors: Adèle
authors: A
authors: van Houwelingen
authors: Margriet
authors: M
authors: Roelse
authors: Saurabh K
authors: SK
authors: Srivastava
authors: Willem
authors: W
authors: Haasnoot
authors: Maarten A
authors: MA
authors: Jongsma
authors: Arend
authors: A
authors: Kolk
authors: Han
authors: H
authors: Zuilhof
authors: Jules
authors: J
authors: Beekwilder
mesh_terms: D000818:Animals; D000907:Antibodies, Bacterial / Q000096:biosynthesis / Q000276:immunology*; D000942:Antigens, Bacterial / Q000276:immunology; D002161:Camelids, New World / Q000276:immunology; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D006360:Heat-Shock Proteins / Q000276:immunology*; D008969:Molecular Sequence Data; D009169:Mycobacterium tuberculosis / Q000276:immunology*; D012680:Sensitivity and Specificity; D014376:Tuberculosis / Q000175:diagnosis* / Q000382:microbiology
chemical_list: D000907:Antibodies, Bacterial; D000942:Antigens, Bacterial; D006360:Heat-Shock Proteins