Abstract
BackgroundThe 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 FindingsTo 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/SignificanceThe 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.
Highlights
Tuberculosis (TB), caused by Mycobacterium tuberculosis (M.tb.), is one of the most prevalent and serious infectious diseases worldwide [1]
Group 1 consists of antibodies binding to three different epitopes (A-23), group 2 those binding to two different epitopes (A-44, A-50, A-89 and B-A1), and group 3 those binding to a single epitope (B-D8, D-F9 and B-F10)
All antibodies positively selected on purified antigen by a direct phage display selection procedure recognized peptide #11, whereas most of the ones negatively selected via a depletion strategy recognized other epitopes, which may be more selective
Summary
Tuberculosis (TB), caused by Mycobacterium tuberculosis (M.tb.), is one of the most prevalent and serious infectious diseases worldwide [1]. With problems like multiple drug resistance (MDR), treatment of diagnosed TB cases is becoming more and more difficult and challenging [3]. TB cases are often intensified due to malnutrition and other allied infections that decrease body immunity, like HIV, especially in the developing parts of the world [4,5]. Detection of TB guarantees fast treatment and can offer much better prognosis. Development of techniques for early and accurate detection iscalled for [6,7]. The 16 kDa heat shock protein (HSP) is an immuno-dominant antigen, used in diagnosis of infectious Mycobacterium tuberculosis (M.tb.) causing tuberculosis (TB). A broad set of twelve 16 kDa specific heavy chain llama antibodies (VHH) has been isolated, and their utility for diagnostic applications was explored
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
