Kidney Week

Abstract: PO1997

Cloning of an IgG Autoantibody Specific for Phospholipase A2 Receptor (PLA2R) Using IgG-Producing Cells from a Patient with Membranous Nephropathy

Session Information

• Podocyte Biology
  October 22, 2020 | Location: On-Demand
  Abstract Time: 10:00 AM - 12:00 PM

Category: Glomerular Diseases

• 1204 Podocyte Biology

Authors

• Huang, Zhi qiang, university of alabama at birmingham, Birmingham, Alabama, United States

Zhi qiang Huang,

• King, Rodney G., university of alabama at birmingham, Birmingham, Alabama, United States

Rodney G. King,

• Knoppova, Barbora, university of alabama at birmingham, Birmingham, Alabama, United States

Barbora Knoppova,

• Reily, Colin, university of alabama at birmingham, Birmingham, Alabama, United States

Colin Reily,

• Bian, Qi, university of alabama at birmingham, Birmingham, Alabama, United States

Qi Bian,

• Hall, Stacy D., university of alabama at birmingham, Birmingham, Alabama, United States

Stacy D. Hall,

• Salant, David J., Boston University Medical Center, Boston, Massachusetts, United States

David J. Salant,

• Beck, Laurence H., Boston University Medical Center, Boston, Massachusetts, United States

Laurence H. Beck,

• Julian, Bruce A., university of alabama at birmingham, Birmingham, Alabama, United States

Bruce A. Julian,

• Novak, Jan, university of alabama at birmingham, Birmingham, Alabama, United States

Jan Novak,

Background

Primary membranous nephropathy (MN) is a common autoimmune kidney disease, in which 70% of patients exhibit circulating autoantibodies to one or more conformational epitopes in PLA2R. Anti-B cell therapies have proved effective to decrease autoantibody production and limit further development of the glomerular subepithelial immune deposits of PLA2R and IgG. However, better characterization of anti-PLA2R autoantibodies is needed.

Methods

We used Epstein-Barr virus (EBV)-immortalized B cells isolated from peripheral blood from an anti-PLA2R seropositive MN patient to develop a protocol for the cloning and expression of recombinant IgG (rIgG) specific for PLA2R. A C-terminally His-tagged fragment consisting of the first 5 domains of human PLA2R was expressed in Expi293 system and affinity purified. B cells were enriched by incubation with biotin labeled reagents (recombinant PLA2R or IgG-specific antibody) and streptavidin-conjugated magnetic beads. A FITC-conjugated N-terminal PLA2R peptide was used to isolate B cells with antigen-specific B-cell receptor (membrane IgG) by FACS. Variable segments of IgG heavy (VH) and light (VL) chains were cloned through a single-cell workflow that allowed expression of rIgG for follow-up screening using an in-house ELISA to assess IgG binding to human PLA2R. Sera from anti-PLA2R seropositive MN patients and healthy controls served as positive and negative controls, respectively.

Results

Starting with 1x10⁸ EBV-immortalized cells, we first isolated 3.6x10⁵ cells with membrane IgG specifically reactive with non-reduced PLA2R. This subpopulation was next stained with FITC-conjugated PLA2R peptide and the corresponding VH and VL of IgG were cloned using our single-cell workflow. We expressed the cloned VH and VL as rIgG in Expi293 system. ELISA confirmed that the rIgG bound PLA2R.

Conclusion

This is the first report of cloning a PLA2R-specific IgG autoantibody from a patient with MN. These approaches can be used for further characterization of the molecular mechanisms of autoimmunity and epitope spreading in MN.

Funding

• NIDDK Support