SuperNAT: A Novel Sensitive Assay for Detection of Low-Abundance Nephrin Autoantibodies (IgG and IgM) and Exploration of Reactive Epitopes in MCD and FSGS

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SuperNAT: A Novel Sensitive Assay for Detection of Low-Abundance Nephrin Autoantibodies (IgG and IgM) and Exploration of Reactive Epitopes in MCD and FSGS

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Co-author 1

Yan Yue yanyue@allograftdx.com AlloDx Biotech Shanghai Co.,Ltd Medicine department Shanghai China -

Co-author 2

Haider Cuello Garcia hecuello@stmail.ujs.edu.cn 1. AlloDx Biotech Shanghai Co.,Ltd 2. School of Medcine, Jiangsu university, Zhenjiang, Jiangsu, China Medicine department Shanghai China -

Co-author 3

Changlin Cao NA AlloDx Biotech Shanghai Co.,Ltd Bioinformatic department Shanghai China -

Co-author 4

Abdelhak Ouzaouit 5102230329@stmail.ujs.edu.cn 1. AlloDx Biotech Shanghai Co.,Ltd. 2. School of Medcine, Jiangsu university, Zhenjiang, Jiangsu, China Medicine department Shanghai China -

Co-author 5

Haitao Liu liuhaitao@allograftdx.com AlloDx Biotech Shanghai Co.,Ltd Medicine department Shanghai China -

Co-author 6

Tingya Jiang jiangtingya@allograftdx.com AlloDx Biotech Shanghai Co.,Ltd Medicine department Shanghai China *

Co-author 7

Yang Zhou zhouyang@ujs.edu.cn School of Medcine, Jiangsu university, Zhenjiang, Jiangsu, China Medicine department Zhenjiang China -

Co-author 8

Haifeng Shi NA 1. AlloDx Biotech Shanghai Co.,Ltd 2. School of Medcine, Jiangsu university, Zhenjiang, Jiangsu, China Medicine department Shanghai China -

Co-author 9

Co-author 10

Co-author 11

Co-author 12

Co-author 13

Co-author 14

Co-author 15

Introduction

The diagnosis of Minimal Change Disease (MCD) and Focal Segmental Glomerulosclerosis (FSGS) remains challenging and still relies primarily on biopsy. Conventional Enzyme Linked ImmunoSorbent Assay(ELISA) struggles to detect low-abundance serum nephrin antibodies due to the interference of blood protein and the false-positive issues for nephrin IgG. Howerver, the IgG immunoprecipitation-enhanced ELISAcannot detect IgM. Moreover, the binding capacity of Nephrin antibodies to different epitopes remains unexplored. Therefore, this study developed a novel Nephrin antibody detection approach called Super Nephrin Antibody Trap (SuperNAT) a high throughput assay to detect both IgG and IgM with automated equipment.

Methods

The experiment carried out using serum samples from 117 healthy individuals and 40 patients with MCD or post-transplant FSGS. In SuperNATthe streptavidin magnetic beads are usedto bind the Biotin-conjugated recombinant Nephrin antigen, and the autoantibodies trapped from the diluted patient serum are detected byanti-human IgG or IgM secondary antibodies . Moreover, antigen-free well serves as the control for each sample.. The SuperNAT assay resists the interference of triglycerides (1.7–5.6 mM) and erythrocytes (0.1–1.0%) to a Nephrin polyantibody (Proteintech). The stability of the conjugates of tbeads and Nephrin antigen were examined in five different preserve solutions at 4℃ up to 3 months. Linear epitopes were predicted bioinformatically (Bepipred 3.0, AlphaFold, DiscoTope 3.0). Six peptides with high-confidence were synthesized and validated using Nephrin IgG-positive samples (n=23) and controls (n=6). We also expressed 9 nephrin extracellular domains in E. coli with C-terminal His-tags

Results

SuperNAT assay revealed Nephrin antibodies level in healthy control had a skewed distribution (coefficient is 1.38, mode is 2.61

Conclusion

SuperNAT is a highly sensitive method for the detection of both IgG and IgM with automation equipment. IgM testing may be warranted in IgG-negative patients. The identified epitopes may correlate with disease progression, warranting further investigation. Future work may employ epitope mapping to correlate nephrin-IgG binding patterns with disease severity in patient cohorts.

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