Method for Depletion of IgG and IgM from Human Serum as Naive Complement Source

Methods Mol Biol. 2021;2227:21-32. doi: 10.1007/978-1-0716-1016-9_2.

Abstract

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.

Keywords: AH50, AP50, CH50; Affinity chromatography; Antibody therapy; Classical pathway; Complement; IgG depletion; IgM depletion; Immunoassay.

MeSH terms

  • Animals
  • Blood Component Removal / methods*
  • Chromatography, Liquid / methods
  • Complement Hemolytic Activity Assay / methods
  • Complement System Proteins / isolation & purification*
  • Complement System Proteins / metabolism
  • Enzyme-Linked Immunosorbent Assay / methods
  • Humans
  • Immunoglobulin G / blood
  • Immunoglobulin G / isolation & purification*
  • Immunoglobulin M / blood
  • Immunoglobulin M / isolation & purification*
  • Mice
  • Rabbits
  • Sheep

Substances

  • Immunoglobulin G
  • Immunoglobulin M
  • Complement System Proteins