Plasma IgG aggregates as biomarkers for multiple sclerosis

Abstract

We recently reported that multiple sclerosis (MS) plasma contains IgG aggregates and induces complement-dependent neuronal cytotoxicity (Zhou et al., 2023). Using ELISA, we report herein that plasma IgG levels in the aggregates can be used as biomarkers for MS. We enriched the IgG aggregates from samples of two cohorts (190 MS and 160 controls) by collecting flow-through after plasma binding to Protein A followed by detection of IgG subclass. We show that there are significantly higher levels of IgG1, IgG3, and total IgG antibodies in MS IgG aggregates, with an AUC >90%; higher levels of IgG1 distinguish secondary progressive MS from relapsing-remitting MS (AUC = 91%). Significantly, we provided the biological rationale for MS plasma IgG biomarkers by demonstrating the strong correlation between IgG antibodies and IgG aggregate-induced neuronal cytotoxicity. These non-invasive, simple IgG-based blood ELISA assays can be adapted into clinical practice for diagnosing MS and SPMS and monitoring treatment responses.

Keywords: Antibody; Biomarker; Cytotoxicity; Diagnosis; IgG; IgG aggregates; IgG1; Immunoglobulin; Multiple sclerosis; Plasma; Progressive MS; SPMS.

Fig. 1.

Significantly higher levels of total protein and IgG antibodies were detected in MS plasma Protein A flow-through (A-FT). A-C. Detection of higher levels of MS plasma IgG is assay-dependent. Plasma (1:200) was added to ELISA plates coated with Protein A (A), Protein G (B), and anti-IgG (Fc) antibodies, followed by incubation with anti-IgG-Fc-Biotinylated antibodies and NeutrAvidin-HRP. A. No differences in IgG levels were detected between MS and controls in Protein A coated plates (n = 10 for SPMS, n = 29 for RRMS, n = 31 for combined controls of HC and OND). B. In Protein G coated plates, higher IgG was detected in RRMS (n = 10 for SPMS, n = 29 for RRMS, n = 30 for combined controls; p < 0.0001). C. ELISA with anti-Fc coated plates showed higher IgG in both SPMS and controls compared to RRMS (n = 8 for SPMS, n = 8 for RRMS, n = 32 for Ctrl; *p = 0.037, ***p = 0.0009, ****p < 0.0001).

Fig. 2.

In the discovery cohort, higher levels of IgG1 and IgG3 were detected in MS A-FT with both commercial kit and in-house IgG subclass ELISA. A-D. Commercial Kit ELISA results for IgG1 and IgG3. Sample numbers are listed in parentheses under each category. A. Kit ELISA showed significantly higher levels of IgG1 in MS A-FT compared to HC (p < 0.001) and NIC (p < 0.0001) but not to IC (p = 0.054). B. ROC curve analysis demonstrating plasma IgG1 as a biomarker separating MS from all other controls (AUC = 0.76; MS = 95, controls = 81). C. Kit ELISA revealed higher levels of IgG3 in MS A-FT compared to HC and NIC controls (both p < 0.0001) and IC (p < 0.05). D. ROC curve analysis of IgG3 as biomarker separating MS from all other controls (AUC = 0.84; MS = 95, controls = 81). E-H, in-house ELISA shows comparable results of higher IgG1 and IgG3 in MS as biomarkers. E. In-house ELISA of IgG1. MS IgG1 is significantly higher than IC (p < 0.001). F. ROC curve data showing IgG1 as a biomarker for MS (AUC = 0.87; MS = 100, controls = 80). G. In-house ELISA of IgG3. H. ROC curve results for IgG3 as MS biomarker (AUC = 0.86; MS = 100, controls = 80).

Fig. 3.

Confirmatory cohort data validate higher IgG1, IgG3, and total IgG as MS biomarkers. A-C. In-house ELISA confirms IgG1, IgG3, and total IgG as MS biomarkers. Results show MS A-FT had significantly higher IgG1, IgG3, and total IgG levels than in controls (p < 0.0001). D–F. ROC curves for respective markers.

Fig. 4.

A moderately strong correlation between MS IgG antibodies and its neuronal cytotoxicity. A. MS has a strong correlation between IgG antibodies and neuronal apoptosis (r = 0.539, p < 0.0001) which may reflect the high N values. B–C. No such associations were found in OND (B) and HC A-FT (C).

Fig. 5.

The IgG1 levels of large IgG aggregates in plasma A-FT can be used to separate SPMS from other subtypes. Plasma IgG1 as a biomarker distinguishing SPMS from RRMS and PPMS. A. Higher levels of IgG1 were detected in SPMS compared to RRMS (p < 0.0001) and PPMS (p < 0.001). Note: Sample numbers are listed under each subtype. B. ROC curve analysis displaying IgG1 as a biomarker distinguishing SPMS from RRMS (AUC = 0.92; SPMS = 40, RRMS = 50). C. ROC curve analysis for IgG1 as plasma biomarker differentiating SPMS from PPMS (AUC = 0.91; SPMS = 40, PPMS = 28). D. SPMS A-FT had significantly higher levels of neuronal cytotoxicity compared to RRMS and PPMS A-FT (p < 0.0001), while RRMS and PPMS A-FT had no significant differences in their neurotoxicity (p = 0.81). E-G. Detection of higher levels of plasma IgG antibodies in SPMS compared to other subtypes is assay-dependent. E. No differences in IgG were detected among MS subtypes with anti-IgG (H + L) capture ELISA. The captured plasma IgG was detected with biotinylated goat anti-human IgG-Fc. F. ELISA with anti-IgG (Fc) coating showed a slight difference between SPMS and RRMS. The captured IgG was detected with biotinylated goat anti-human IgG (H + L). No differences were detected between SPMS and PPMS; however, SPMS had higher total IgG than RRMS (p < 0.05). G. Direct coating ELISA showed significantly higher levels of total IgG in SPMS. Plasma (1:200) was coated directly onto ELISA plate wells, followed by HRP-goat anti-human IgG (H + L) detection. SPMS had higher levels of total IgG compared to RRMS (p < 0.05) and PPMS (p < 0.01).

Fig. 6.

Relationship between age/gender and plasma IgG antibodies in MS. A. There is a positive correlation between age and IgG1 in MS patients (r = 0.29, p < 0.0001, n = 190). B. A positive correlation exists between age and IgG3 in MS (r = 0.21, p = 0.004, n = 190). C–D. No relationships were observed between age and IgG1/IgG3 in controls (n = 160). E-F. No gender differences were found in MS (E) and controls (F). MS = 190, control = 160.