title: 16 kDa heat shock protein from heat-inactivated Mycobacterium tuberculosis is a homodimer - suitability for diagnostic applications with specific llama VHH monoclonals.
abstract: 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.
  
  
  RESULTS
  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
  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.
authors.affiliation: Plant Research International, Wageningen, The Netherlands.
authors.forename: Saurabh K
authors.initials: SK
authors.lastname: Srivastava
authors.forename: Vincent J B
authors.initials: VJ
authors.lastname: Ruigrok
authors.forename: Natalie J
authors.initials: NJ
authors.lastname: Thompson
authors.forename: Anke K
authors.initials: AK
authors.lastname: Trilling
authors.forename: Albert J R
authors.initials: AJ
authors.lastname: Heck
authors.forename: Cees
authors.initials: C
authors.lastname: van Rijn
authors.forename: Jules
authors.initials: J
authors.lastname: Beekwilder
authors.forename: Maarten A
authors.initials: MA
authors.lastname: Jongsma
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000918:Antibody Specificity; D001426:Bacterial Proteins / Q000737:chemistry / Q000276:immunology / Q000378:metabolism*; D002161:Camelids, New World; D018604:Epitope Mapping; D006360:Heat-Shock Proteins / Q000737:chemistry / Q000276:immunology / Q000378:metabolism*; D006358:Hot Temperature; D007143:Immunoglobulin Heavy Chains / Q000276:immunology*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009169:Mycobacterium tuberculosis / Q000276:immunology / Q000302:isolation & purification* / Q000378:metabolism / Q000502:physiology; D010446:Peptide Fragments / Q000737:chemistry / Q000276:immunology; D055503:Protein Multimerization; D020836:Protein Structure, Quaternary; D013183:Sputum / Q000382:microbiology; D014376:Tuberculosis / Q000175:diagnosis
chemical_list: D001426:Bacterial Proteins; D006360:Heat-Shock Proteins; D007143:Immunoglobulin Heavy Chains; D010446:Peptide Fragments