<i>M. tuberculosis</i> binding VHH antibody fragments.

<p>Protein sequence of 62 selected VHH antibody fragments selected by phage display for binging to <i>M. tuberculosis</i>. Dots indicate sequence identity, and dashes indicate gaps. The three complementarity determining regions CDR1, CDR2 and CDR3 are shaded. Characteristic VH-VHH hallmark substitutions (Leu12Ser, Val42Phe or Val42Tyr, Gly49Glu, Leu50Arg or Leu50Cys and Trp52Gly or Trp52Leu (the last substitution is less well conserved) (the ImMunoGeneTics system <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0026754#pone.0026754-Lefranc1" target="_blank">[52]</a> was followed) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0026754#pone.0026754-Muyldermans2" target="_blank">[15]</a> are underlined. The 12 clones selected for further investigations are underlined. VHH protein sequences labeled A-x (with x = 1–96) resulted from direct selection using semi-purified protein antigen, protein sequences labeled B-yx (with y = A–F and x = 1–12) were achieved by depletion method.</p

Western blot to discover antigen of VHHs.

<p>A) Western blot using VHH A-23 as a probe. 9 µg <i>M. tb 1</i> lysate were run on a 15% SDS-PAGE gel in lanes A–D, and transferred to a nitrocellulose membrane. Lane A: incubated with VHH A-23 and detected using anti-VSV-HRP; Lane B: incubated with VHH A-23 and detected using anti-HIS-HRP; Lane C: incubated with detection antibody anti-VSV-HRP; Lane D: incubated with detection antibody anti-HIS-HRP. B) Western blot analysis to confirm the specificity of VHH A-23. Lane 1: 9 µg <i>M. tb 22</i> lysate detected by monoclonal mouse 16 kDa antibody, using anti-mouse-HRP secondary antibody; Lane 2: 9 µg <i>M. tb 22</i> lysate detected by VHH A-23, using anti-VSV-HRP secondary antibody. Lane 3: 3 µg of purified recombinant 16 kDa protein detected by VHH A-23, using anti-VSV-HRP secondary antibody. Due to the tags added for purification and detection purposes the calculated mass of the recombinant 16 kDa protein is 21 kDa. Indicated are the positions of relevant size markers in kDa. C) Western blot analysis of native PAGE analysis. Lane 1: 5 µg <i>M. tb 27</i> lysate; Lane 2: 1 µg of purified recombinant 16 kDa protein. Detection was by VHH A-23, using anti-VSV-HRP secondary antibody.</p

Direct ELISA to confirm specific binging of VHH antibody fragments to tuberculosis lysate.

<p>VHH antibody fragments A-23 and B-F10 with A) <i>M. tuberculosis</i> whole cells, <i>M. tuberculosis</i> cell lysate and media in which <i>M. tuberculosis</i> bacteria were grown. B) different <i>M. tuberculosis</i> lysates as well as lysates of <i>M. bcg</i>, <i>M. avium</i>, <i>M. kansasii</i>, <i>M. smegmatis, S. pneumoniae and H. influenzae</i>. Measurements were performed in duplicate, expressed as means ± SD.</p