Abstract: FR-OR87
Host-Cell Line Origin of Recombinant Human Nephrin Impacts Anti-nephrin Antibody Enzyme-Linked Immunosorbent Assay (ELISA): Insights into Glycan-Mediated Discrepancy
Session Information
- Pathology and Lab Medicine Advances
October 25, 2024 | Location: Room 2, Convention Center
Abstract Time: 05:20 PM - 05:30 PM
Category: Pathology and Lab Medicine
- 1800 Pathology and Lab Medicine
Authors
- Ghasemi, Maryam, Brigham and Women's Hospital, Boston, Massachusetts, United States
- Weins, Astrid, Brigham and Women's Hospital, Boston, Massachusetts, United States
- Watts, Andrew James Baxter, Brigham and Women's Hospital, Boston, Massachusetts, United States
Background
We identified circulating autoantibodies targeting the extracellular domain (ECD) of human nephrin in a subset of patients with minimal change disease. We observed a discrepancy in ELISA results when utilizing human nephrin ECD expressed in a human versus a murine cell line. Nephrin is glycosylated and notably this murine cell line expresses two immunogenic glycans, galactose-α-1,3-galactose (α-Gal) and N-glycolylneuramic acid (Neu5Gc), which are not expressed in human cells. Most human sera contain immunoglobulin G (IgG) against α-Gal and Neu5Gc, which we hypothesized may account for the observed difference in the IgG binding.
Methods
Indirect ELISA for anti-nephrin antibodies in sera was conducted as previously described using affinity-purified recombinant human nephrin ECD (>90% purity) expressed in either human embryonic kidney (HEK) or non-secreting murine myeloma (NSO) cell lines, purchased from Sino Biological (Cat. 17757-H08H) and R&D systems (Cat. 9399-NN) respectively. The amino acid sequence was similar. α-Gal and Neu5Gc expression on the nephrin ECD were evaluated by ELISA. Serum was pre-incubated with porcine RBCs, which express α-Gal and Neu5Gc, and IgG binding to nephrin was evaluated pre- and post-incubation.
Results
Using healthy control sera, we observed more IgG binding to the human nephrin expressed in the NSO compared with the HEK cell line (Fig. 1). We confirmed α-Gal and Neu5Gc presence only on the NSO-expressed human nephrin. Pre-incubation of the sera with porcine RBCs abrogated this higher IgG binding with NSO-expressed nephrin to a level observed with HEK-expressed nephrin (Fig. 2)
Conclusion
We observed more IgG binding from healthy control sera to human nephrin ECD expressed in a murine NSO cell line compared with a human HEK cell line, and this was abrogated by pre-incubating the sera with porcine RBCs. This suggests that IgG binding to α-Gal and Neu5Gc may explain this difference. We therefore recommend using recombinant human nephrin specifically expressed in human cell lines when evaluating patient sera for nephrin autoantibodies by ELISA to minimize false positives.
Funding
- NIDDK Support