Hydrogen–Deuterium Exchange Epitope Mapping Reveals Distinct Neutralizing Mechanisms for Two Monoclonal Antibodies against Diphtheria Toxin

Abstract

Mass spectra of the peptides corresponding to the epitopes, positive and negative controls of ForteBio binding studies, binding rate comparison of mAb 2−25 binding to DTx and to DTx/HBEGF complex, and Western blot analysis of the mAbs binding to full-length and partially hydrolyzed DTx (PDF)

Genetically detoxified pertussis toxin displays near identical structure to its wild-type and exhibits robust immunogenicity

“…HDX-MS experiments were performed using the LEAP PAL automation technology coupled to Waters ultra-performance liquid chromatography (UPLC) and Synapt G2-S MS system as previously reported with minor modifications [44][45][46] . A detailed experimental workflow has been described elsewhere and only modifications were described here 46 . For non-deuterated and deuterated experiments, antigens were diluted in 10 mM potassium phosphate buffer (Sigma-Aldrich), pH 7.5, and deuteration buffer (10 mM potassium phosphate (Sigma-Aldrich), pD 7.5, respectively.…”

Hydrogen-deuterium exchange mass spectrometry reveals three unique binding responses of mAbs directed to the catalytic domain of hCAIX

“…This method is a powerful tool for monitoring changes in the average conformational state of proteins, and is routinely applied in the characterization of antibody-antigen interactions. 48 , 53 To facilitate the HDX-MS interrogation we expressed a monomeric form of hCAIX that is devoid of its PG domain by removing the inter-dimer disulfide bond at Cys 38 ( Figure 1 , ΔPG-hCAIX Cys38Ser ). 12 This construct maintained catalytic activity and structural integrity and allowed for a simplified HDX-MS experimental design to be implemented.…”

Epitope Screening Using Hydrogen/Deuterium Exchange Mass Spectrometry (HDX-MS): An Optimized Workflow for Accelerated Evaluation of Lead Monoclonal Antibodies

“…The HDX experiments were performed as previously described with few modifications. [16,25] For antigen-only HDX experiments, 15 μM of PRN was used and for the complex, equimolar of 15 μM of PRN and mAb were incubated in buffer E (10 mM potassium phosphate buffer pH 7.5). 7.5 μL of the sample was mixed with 32.5 μL of deuterated buffer, buffer L (10 mM potassium phosphate pD 7.5) at 25°C.…”