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Method for Depletion of IgG and IgM from Human Serum as Naive Complement Source.
Zwarthoff, Seline A; Magnoni, Simone; Aerts, Piet C; van Kessel, Kok P M; Rooijakkers, Suzan H M.
Affiliation
  • Zwarthoff SA; Medical Microbiology, University Medical Center Utrecht, Utrecht University, Utrecht, The Netherlands.
  • Magnoni S; Medical Microbiology, University Medical Center Utrecht, Utrecht University, Utrecht, The Netherlands.
  • Aerts PC; Medical Microbiology, University Medical Center Utrecht, Utrecht University, Utrecht, The Netherlands.
  • van Kessel KPM; Medical Microbiology, University Medical Center Utrecht, Utrecht University, Utrecht, The Netherlands. k.kessel@umcutrecht.nl.
  • Rooijakkers SHM; Medical Microbiology, University Medical Center Utrecht, Utrecht University, Utrecht, The Netherlands.
Methods Mol Biol ; 2227: 21-32, 2021.
Article in En | MEDLINE | ID: mdl-33847927
Understanding how human complement proteins interact with human antibodies is important for the development of antibody therapies and understanding autoimmune diseases. At present, many groups use baby rabbit serum as a source of complement because, in contrast to human serum, it lacks preexisting antibodies. However, for characterization of human (monoclonal) antibodies, human serum would be a preferred source of complement. To prevent complement activation via naturally occurring antibodies, this human serum ideally lacks IgG and IgM. Here we describe how to deplete human serum of naturally occurring IgG and IgM using fast protein liquid affinity chromatography (FPLC) while minimizing the loss of serum complement activity. We also describe assays that can be used to validate depletion of IgG and IgM (IgG, IgM, and C1q sandwich ELISAs) and functionally assess remaining serum complement activity (hemolytic assays CH50 and AH50). Finally, we demonstrate how captured IgG and IgM can be purified.
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Full text: 1 Collection: 01-internacional Database: MEDLINE Main subject: Blood Component Removal / Complement System Proteins / Immunoglobulin G / Immunoglobulin M Limits: Animals / Humans Language: En Journal: Methods Mol Biol Year: 2021 Document type: Article

Full text: 1 Collection: 01-internacional Database: MEDLINE Main subject: Blood Component Removal / Complement System Proteins / Immunoglobulin G / Immunoglobulin M Limits: Animals / Humans Language: En Journal: Methods Mol Biol Year: 2021 Document type: Article