A broad set of different llama antibodies specific for a 16 kDa heat shock protein of Mycobacterium tuberculosis |
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Authors: | Trilling Anke K de Ronde Hans Noteboom Linda van Houwelingen Adèle Roelse Margriet Srivastava Saurabh K Haasnoot Willem Jongsma Maarten A Kolk Arend Zuilhof Han Beekwilder Jules |
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Affiliation: | 1Plant Research International, Wageningen, The Netherlands;2Laboratory of Organic Chemistry, Wageningen University, Wageningen, The Netherlands;3Royal Tropical Institute, Amsterdam, The Netherlands;4Van ''t Hoff Institute for Molecular Sciences, University of Amsterdam, Amsterdam, The Netherlands;5RIKILT-Institute of Food Safety, Wageningen, The Netherlands;Statens Serum Institute, Denmark |
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Abstract: | BackgroundRecombinant 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.Methodology/Principal FindingsAntibodies 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/SignificanceA 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. |
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