Fig. 4: Characterisation of anti-TPI mAbs.
From: Epitope-specific immunity against Staphylococcus aureus coproporphyrinogen III oxidase
a Binding domain of H8 / F3 mAbs (red) was identified by western blot analysis using recombinant His-, or Strep-tagged TPI-fragments and classified as detectable (+) or non-detectable (−) by H8/F3 mAbs in comparison to anti-Strep-tag antibodies. b S. aureus TPI structure (3m9y.pdb) was visualised by EzMol2.1 with the identified aa of the H8/F3 binding domain in red. c Epitopes of mAb C4 (green) and mAb C8 (blue) were identified by PepSpot technology using overlapping 15mer TPI peptides with overlapping sequences of 11 aa. d Competition ELISA. Binding of DyLight-649-conjugated anti-TPI mAb H8 to recombinant rTPI coated on ELISA MaxiSorp plate, was competed with unconjugated, indicated mAbs. Binding was determined by fluorescence measurement (Ex 646/Em 674). Data are presented as mean ± s.d. (n = 2). e Saturation binding curve was generated by plotting absorbance signals (OD450nm) of increasing amounts of anti-TPI huMAb H8 to rTPI coated on ELISA MaxiSorp plate using the GraphPadPrism 8.4 software. Kd was calculated by non-linear fitting and the equation for one-site binding model [Y = Bmax*X/(Kd + X)].
