Abstract: TH-PO576
Expanding the Use of Surface Plasmon Resonance (SPR) for Characterizing Nephritic Factors in Patients with C3 Glomerulopathy (C3G)
Session Information
- Glomerular Diseases: Omics, Biomarkers, and Tools
October 24, 2024 | Location: Exhibit Hall, Convention Center
Abstract Time: 10:00 AM - 12:00 PM
Category: Glomerular Diseases
- 1401 Glomerular Diseases: Mechanisms, including Podocyte Biology
Authors
- Culek, Christopher, University of Iowa Hospitals and Clinics, Iowa City, Iowa, United States
- Fierce, Cecelia F., University of Iowa Hospitals and Clinics, Iowa City, Iowa, United States
- Nester, Carla M., University of Iowa Hospitals and Clinics, Iowa City, Iowa, United States
Group or Team Name
- Molecular Otolaryngology and Renal Research Laboratories.
Background
Nephritic factors (Nefs) are convertase-directed autoantibodies present in up to 80% of C3 glomerulopathy patients.1 Despite this prevalence and the associated complement dysregulation, there remains much to be learned about Nef characteristics. Previous work used SPR to show Nefs drive disease by stabilizing the convertase and amplifying complement activity.2 However, this work was unable to characterize patients with Nefs sensitive to regulation by DAF or FH (scenarios evident in our patient population). This deficiency limits our understanding of individual patients and opens the possibility that we are missing populations of Nefs all together. We sought to develop a simple regulator-independent SPR technique that can provide a reproducible half-life of the Nef-convertase interaction.
Methods
C3b was immobilized to an SPR microfluidic chip at 1200RUs. Several concentrations of FB, FD, and IgG were co-injected onto the C3b ligand to form convertase-IgG complexes. The complex dissociated at 37C for 600 seconds (~6-7 half-lives of reagent convertase), to allow total decay of unbound convertase complexes. Then the sensorgram was normalized to 100RUs. The estimated half-life was calculated as the time at which the sensorgram reached 50RUs remaining. We evaluated nine replicates of one polyclonal Nef-positive IgG test subject and five Nef-negative test subjects.
Results
Both the Nef-positive and Nef-negative polyclonal IgG show specificity to the convertase, as previously supported.3 The Nef-positive stabilization half-life ranged from 2072 to 2084 seconds with a mean of 2080, while the Nef-negative stabilization half-lives ranged from 464 to 598 seconds. The Nef-positive sample had low variability with a SD of only 4.609, and a SE of 1.537. The estimated half-life was independent of the concentration of FB or FD.
Conclusion
This data suggests our approach was able to reproducibly provide an estimated half-life for the Nef-convertase complex. Furthermore, it shows distinct kinetics between the Nef-positive and Nef-negative autoantibodies. The assay expands the scope of current Nef evaluation by removing the requirement of complement regulator proteins in the kinetic analysis and provides valuable kinetic information for evaluating complement dysregulation in Nef-positive C3G patients regardless of regulator sensitivity.
Citations
1. PMC3280037
2. PMC3608896
3. PMC10695638
Funding
- NIDDK Support