PMID- 31681303
OWN - NLM
STAT- MEDLINE
DCOM- 20201109
LR  - 20201109
IS  - 1664-3224 (Electronic)
IS  - 1664-3224 (Linking)
VI  - 10
DP  - 2019
TI  - Low pH Exposure During Immunoglobulin G Purification Methods Results in 
      Aggregates That Avidly Bind Fcgamma Receptors: Implications for Measuring Fc 
      Dependent Antibody Functions.
PG  - 2415
LID - 10.3389/fimmu.2019.02415 [doi]
LID - 2415
AB  - Evaluating the biophysical and functional nature of IgG is key to defining 
      correlates of protection in infectious disease, and autoimmunity research 
      cohorts, as well as vaccine efficacy trials. These studies often require small 
      quantities of IgG to be purified from plasma for downstream analysis with high 
      throughput immunoaffinity formats which elute IgG at low-pH, such as Protein G 
      and Protein A. Herein we sought to compare Protein G purification of IgG with an 
      immunoaffinity method which elutes at physiological pH (Melon Gel). Critical 
      factors impacting Fc functionality with the potential to significantly influence 
      FcgammaR binding, such as IgG subclass distribution, N-glycosylation, aggregation, 
      and IgG conformational changes were investigated and compared. We observed that 
      transient exposure of IgG to the low-pH elution buffer, used during the Protein G 
      purification process, artificially enhanced recognition of Fcgamma Receptors (FcgammaRs) 
      as demonstrated by Surface Plasmon Resonance (SPR), FcgammaR dimer ELISA, and a 
      functional cell-based assay. Furthermore, low-pH exposed IgG caused 
      conformational changes resulting in increased aggregation and hydrophobicity; 
      factors likely to contribute to the observed enhanced interaction with FcgammaRs. 
      These results highlight that methods employed to purify IgG can significantly 
      alter FcgammaR-binding behavior and biological activity and suggest that the IgG 
      purification approach selected may be a previously overlooked factor contributing 
      to the poor reproducibility across current assays employed to evaluate 
      Fc-mediated antibody effector functions.
CI  - Copyright (c) 2019 Lopez, Scott, Wines, Hogarth, Wheatley, Kent and Chung.
FAU - Lopez, Ester
AU  - Lopez E
AD  - Department of Microbiology and Immunology, Peter Doherty Institute for Infection 
      and Immunity, University of Melbourne, Parkville, VIC, Australia.
FAU - Scott, Nichollas E
AU  - Scott NE
AD  - Department of Microbiology and Immunology, Peter Doherty Institute for Infection 
      and Immunity, University of Melbourne, Parkville, VIC, Australia.
FAU - Wines, Bruce D
AU  - Wines BD
AD  - Immune Therapies Group, Burnet Institute, Melbourne, VIC, Australia.
AD  - Department of Immunology and Pathology, Monash University, Melbourne, VIC, 
      Australia.
AD  - Department of Clinical Pathology, The University of Melbourne, Melbourne, VIC, 
      Australia.
FAU - Hogarth, P Mark
AU  - Hogarth PM
AD  - Immune Therapies Group, Burnet Institute, Melbourne, VIC, Australia.
AD  - Department of Immunology and Pathology, Monash University, Melbourne, VIC, 
      Australia.
AD  - Department of Clinical Pathology, The University of Melbourne, Melbourne, VIC, 
      Australia.
FAU - Wheatley, Adam K
AU  - Wheatley AK
AD  - Department of Microbiology and Immunology, Peter Doherty Institute for Infection 
      and Immunity, University of Melbourne, Parkville, VIC, Australia.
FAU - Kent, Stephen J
AU  - Kent SJ
AD  - Department of Microbiology and Immunology, Peter Doherty Institute for Infection 
      and Immunity, University of Melbourne, Parkville, VIC, Australia.
AD  - Infectious Diseases Department, Melbourne Sexual Health Centre, Central Clinical 
      School, Alfred Health, Monash University, Melbourne, VIC, Australia.
AD  - ARC Centre of Excellence in Convergent Bio-Nano Science and Technology, 
      University of Melbourne, Parkville, VIC, Australia.
FAU - Chung, Amy W
AU  - Chung AW
AD  - Department of Microbiology and Immunology, Peter Doherty Institute for Infection 
      and Immunity, University of Melbourne, Parkville, VIC, Australia.
LA  - eng
PT  - Journal Article
PT  - Research Support, Non-U.S. Gov't
DEP - 20191011
PL  - Switzerland
TA  - Front Immunol
JT  - Frontiers in immunology
JID - 101560960
RN  - 0 (Immunoglobulin Fc Fragments)
RN  - 0 (Immunoglobulin G)
RN  - 0 (Immunoglobulin Isotypes)
RN  - 0 (Receptors, IgG)
SB  - IM
MH  - Antibody Affinity
MH  - Chromatography, Liquid
MH  - Glycosylation
MH  - Humans
MH  - *Hydrogen-Ion Concentration
MH  - Hydrophobic and Hydrophilic Interactions
MH  - Immunoglobulin Fc Fragments/immunology/*metabolism
MH  - Immunoglobulin G/chemistry/immunology/*isolation & purification/*metabolism
MH  - Immunoglobulin Isotypes
MH  - Kinetics
MH  - Mass Spectrometry
MH  - Phagocytosis
MH  - Protein Binding
MH  - Receptors, IgG/*metabolism
PMC - PMC6797627
OTO - NOTNLM
OT  - Fc functions
OT  - Fcgamma receptors
OT  - IgG purification
OT  - antibody
OT  - antibody dependent cellular phagocytosis (ADCP)
OT  - melon gel
OT  - protein G
EDAT- 2019/11/05 06:00
MHDA- 2020/11/11 06:00
PMCR- 2019/01/01
CRDT- 2019/11/05 06:00
PHST- 2019/07/24 00:00 [received]
PHST- 2019/09/26 00:00 [accepted]
PHST- 2019/11/05 06:00 [entrez]
PHST- 2019/11/05 06:00 [pubmed]
PHST- 2020/11/11 06:00 [medline]
PHST- 2019/01/01 00:00 [pmc-release]
AID - 10.3389/fimmu.2019.02415 [doi]
PST - epublish
SO  - Front Immunol. 2019 Oct 11;10:2415. doi: 10.3389/fimmu.2019.02415. eCollection 
      2019.