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Background: The 16 kDa heat shock protein (HSP) is an immuno-dominant antigen, used in diagnosis of infectious Mycobacterium tuberculosis (M.tb.) causing tuberculosis (TB). Its use in serum-based diagnostics is limited, but for the direct identification of M.tb. bacteria in sputum or cultures it may represent a useful tool. Recently, a broad set of twelve 16 kDa specific heavy chain llama antibodies (VHH) has been isolated, and their utility for diagnostic applications was explored. Methodology/Principal Findings: To identify the epitopes recognized by the nine (randomly selected from a set of twelve 16 kDa specific VHH antibodies) distinct VHH antibodies, 14 overlapping linear epitopes (each 20 amino acid long) were characterized using direct and sandwich ELISA techniques. Seven out of 14 epitopes were recognized by 8 out of 9 VHH antibodies. The two highest affinity binders B-F10 and A-23 were found to bind distinct epitopes. Sandwich ELISA and SPR experiments showed that only B-F10 was suitable as secondary antibody with both B-F10 and A-23 as anchoring antibodies. To explain this behavior, the epitopes were matched to the putative 3D structure model. Electrospray ionization time-of-flight mass spectrometry and size exclusion chromatography were used to determine the higher order conformation. A homodimer model best explained the differential immunological reactivity of A-23 and B-F10 against heat-treated M.tb. lysates. Conclusions/Significance: The concentrations of secreted antigens of M.tb. in sputum are too low for immunological detection and existing kits are only used for identifying M.tb. in cultures. Here we describe how specific combinations of VHH domains could be used to detect the intracellular HSP antigen. Linked to methods of pre-concentrating M.tb. cells prior to lysis, HSP detection may enable the development of protein-based diagnostics of sputum samples and earlier diagnosis of diseases. [ABSTRACT FROM AUTHOR]

Uniform orientation of capture molecules on biosensors has been reported to increase sensitivity. Here it is investigated which analyte properties contribute to sensitivity by orientation. Orientation of capture molecules on biosensors was investigated using variable domains of llama heavy-chain antibodies (VHHs) as capture molecule, and a surface plasmon resonance (SPR) chip as biosensor. Two VHHs were tested in this study: one recognizing foot-and-mouth disease virus (FMDV) and another recognizing the 16 kDa heat-shock protein of Mycobacterium tuberculosis. SPR chips with randomly immobilized biotinylated VHHs were compared to streptavidin-coated SPR chips, on which similar quantities of oriented biotinylated VHHs were non-covalently immobilized. Analytes that differ in molecular weight, epitope number and epitope affinity were compared using the FMDV-recognizing VHH. When binding of intact FMDV particles (146 S; 8200 kDa) or pentameric FMDV coat protein aggregates (12 S; 282 kDa) was detected, a modest (1-2-fold) increase in sensitivity was observed. When a 26-residue peptide (3 kDa) containing the epitope for VHH recognition was tested, much larger effects of capture molecule orientation (14-fold) on signal were observed. A 20-227-fold improvement was also observed when the epitope peptide was covalently linked to bovine serum albumin (67 kDa) or R-phycoerythrin (240 kDa). The results indicate that orientation of the capture molecule hardly affects high-affinity interactions, while it leads to strong improvements in sensitivity for lower-affinity interactions., (Copyright © 2012 Elsevier B.V. All rights reserved.)