Epitope and affinity determination of recombinant Mycobacterium tuberculosis Ag85B antigen towards anti-Ag85 antibodies using proteolytic affinity-mass spectrometry and biosensor analysis

Anal Bioanal Chem. 2019 Jan;411(2):439-448. doi: 10.1007/s00216-018-1466-z. Epub 2018 Nov 29.


Tuberculosis (TB) is the first cause of death from infectious diseases worldwide. Only a single anti-TB vaccine is currently available for clinical use, but its efficacy is not achieved with certainty. The aim of this work is to provide a basis for the rational design of a neo-glycoconjugate vaccine against TB. Structural characterization of recombinant antigenic proteins from Mycobacterium tuberculosis (MTB) Ag85B (rAg85B, variants, and semi-synthetic glycoconjugates) was initially carried out. Identification of antibody epitope analyses by proteolytic affinity-mass spectrometry and surface plasmon resonance (SPR) biosensor analyses were performed in order to qualitatively identify and quantitatively characterize interaction structures of the antigens with antibodies from different sources. A commercial monoclonal antibody and polyclonal antibodies from different sources (patients with active TB, vaccinated individuals, and a healthy control) were employed to analyze antigen-antibody interactions. These combined approaches provided the identification of different assembled epitope regions on the recombinant MTB antigens, their affinity binding constants in the interactions with specific antibodies, and revealed the importance of protection from excessive glycosylation. The identified epitope peptides should constitute a suitable basis for the design of new specific target vaccines. Graphical abstract ᅟ.

Keywords: Affinity mass spectrometry; Ag85B; Antibody epitope identification; Glycoconjugate vaccine; Surface plasmon resonance; Tuberculosis.

MeSH terms

  • Amino Acid Sequence
  • Antibodies, Bacterial*
  • Antibody Affinity*
  • Antigens, Bacterial*
  • Biosensing Techniques
  • Epitopes / chemistry*
  • Mass Spectrometry / methods*
  • Models, Molecular
  • Mycobacterium tuberculosis / metabolism*
  • Protein Conformation
  • Proteolysis


  • Antibodies, Bacterial
  • Antigens, Bacterial
  • Epitopes