Abstract: SA-PO605
THSD7A Knockout Mice as a Valuable Tool for the Generation of Domain-Specific Monoclonal Antibodies
Session Information
- Glomerular Diseases: Immunology, Inflammation - II
November 09, 2019 | Location: Exhibit Hall, Walter E. Washington Convention Center
Abstract Time: 10:00 AM - 12:00 PM
Category: Glomerular Diseases
- 1202 Glomerular Diseases: Immunology and Inflammation
Authors
- Seifert, Larissa, University Medical Center Hamburg-Eppendorf, Hamburg, Germany
- Zahner, Gunther, University Medical Center Hamburg-Eppendorf, Hamburg, Germany
- Meyer-Schwesinger, Catherine, University Medical Center Hamburg-Eppendorf, Hamburg, Germany
- Wiech, Thorsten, University Medical Center Hamburg-Eppendorf, Hamburg, Germany
- Huber, Tobias B., University Medical Center Hamburg-Eppendorf, Hamburg, Germany
- Koch-nolte, Friedrich, University Medical Center Hamburg-Eppendorf, Hamburg, Germany
- Tomas, Nicola M., University Medical Center Hamburg-Eppendorf, Hamburg, Germany
Background
Autoantibodies against multiple domains in THSD7A cause membranous nephropathy (MN). However, the function of THSD7A in podocytes, the precise mechanisms of antibody-induced glomerular injury and the role of the targeted epitopes are largely unknown.
Methods
Embryonic stem cells for generation of THSD7A-ko first mice were purchased from UCDavis KOMP. Since ko first mice were found to contain residual expression of THSD7A in podocytes, mice were further bred to obtain a true constitutive THSD7A ko status (THSD7A-/-).
For the generation of poly- and monoclonal antibodies (MABs), 12w-old THSD7A-/- mice were immunized using the purified murine antigen fragments d1_d2 and d15_d16, corresponding to the domains most frequently recognized by patient autoantibodies. Spleen and lymph nodes were collected 7w after immunization and fused with SP2/0 myeloma cells. Cell clones expressing anti-THSD7A antibodies were selected using an immunofluorescence test on THSD7A-transfected CHO cells. Positive clones were cultured and IgG was purified from cell culture supernatant.
Results
THSD7A-/- mice showed no glomerular expression of THSD7A in immunofluorescence and Western blot. Glomeruli appeared normal in PAS staining and no ultrastructural changes were observed by EM. Mice had no proteinuria by the age of 1y.
Immunization of THSD7A-/- mice induced high levels of domain-specific anti-THSD7A antibodies. Fusion of splenic cells with myeloma cells resulted in 3 clones producing antibodies against THSD7A. One MAB recognized d1_d2 (IgG1) and two MABs recognized d15_d16 (IgG1 and IgG2b). These MABs were suitable for the detection of THSD7A in immunofluorescence, Western and dot blot. Transfer of individual MABs to WT BALB/c mice resulted in glomerular binding of mouse IgG, but no complement deposition and no proteinuria.
Conclusion
In preliminary investigations, THSD7A-/- mice do not show significant glomerular alterations, suggesting that THSD7A expression is not integral for basal glomerular function or that other unidentified molecules can compensate for the lack of THSD7A. THSD7A-/- mice are a powerful tool for the generation of poly- and monoclonal antibodies and these antibodies can potentially be used to dissect the mode of antibody-induced glomerular damage and the role of targeted epitopes in MN.