Figure 1.
Summary of affinity interactions of 14 overlapping peptides derived from the HSP protein with 9 different anti-HSP VHH antibodies.
VHH numbering is according to Trilling et al 2011 [18]. VHHs are indicated above the central portions of the interacting peptides.
Figure 2.
Peptides (14 overlapping 20-mers) representing potential epitopes of the HSP protein mapped against different VHH antibodies in direct ELISA.
Panel A - Scheme applied in B; Panel B - Results of direct ELISA with coated peptides; Panel C - Scheme applied in D; Panel D - Direct ELISA with VHH antibodies coated to the well.
Table 1.
Summary of interactions of 14 peptides derived from the HSP protein with 9 different anti-HSP VHH antibodies [18].
Figure 3.
Comparison of purified recombinant HSP protein with M.tb.-HSP in sandwich ELISA assays with antibody A-23 AVI as capture antibody.
Figure 4.
Plots of “sandwich SPR” showing the binding kinetics of B-F10 or A-23 binding to HSP as a secondary antibody in a sandwich assay in which HSP was first captured by A-23 or B-F10.
Four different sandwich variations are shown: A-23-HSP-A-23 or -B-F10 (top panel) and B-F10-HSP-A-23 or -B-F10 (lower panel).
Table 2.
SPR assay results showing the observed response units (RU) for rHSP-tag (HSP) and the detection antibodies A-23 and B-F10.
Figure 5.
Size exclusion chromatography (SEC) for apparent molecular weight determination of HSP.
Where figure A shows SEC plot of different HSP samples i.e. purified recombinant HSP with tag (rHSP-tag), unpurified recombinant native HSP ( rHSP), heated unpurified recombinant native HSP ( heated-rHSP) and M.tb. lysates (Mtb-HSP) while figure B shows the dot blot of interaction of 16 kDa protein present in these SEC fractions with antibody B-F10, where lane 4&5 shows fractions of different HSP protein eluting at ∼301 kDa, lane 7–9 shows fractions of different HSP protein eluting at ∼55–61 kDa.
Figure 6.
Native ESI-TOF-MS spectrum of HSP confirming absence of oligomers greater than the dimer after urea-based isolation.
Sample complexity at low m/z is due to proteolytic loss of tags prior to purification. The various HSP species are indicated in the spectrum: monomer without tag (light blue circle), monomer with tag (dark blue circle), dimer formed from monomers with tags (double dark blue circles), and dimer formed from mixed monomers (double circles, light and dark blue). Other signals in the spectrum stem from contaminants, including the released tags. Deviations of calculated and measured mass values are by exopeptidases pruning the proteins. Denaturing SDS-PAGE coomassie stained gel (inset) shows two prominent bands at 21 and 17 kDa size corresponding to monomers with and without tags.
Figure 7.
Model showing the arrangement of dimer structure of HSP protein along with the representation of possible sites of VHH antibodies B-F10 and A-23.
Figure 8.
Models explaining the blocked detection by A-23 antibodies.
The diagrams show the capture of HSP dimer with VHH antibodies B-F10 and A-23 and successful detection with B-F10 panels A–B) and failed detection with A-23 (panels C–D).