Quantification of Autoreactive Antibodies in Mice upon Experimental Autoimmune Encephalomyelitis

Summary

Experimental autoimmune encephalomyelitis (EAE) is an animal model of multiple sclerosis (MS), which shares with the human disease a robust humoral autoimmune response. Here, we report a simple and flexible ELISA protocol to quantify autoantibodies in the serum of EAE immunized mice.

Abstract

Experimental autoimmune encephalomyelitis (EAE) is a disease model that recapitulates the autoimmune disorder multiple sclerosis (MS) at histopathological and molecular levels. EAE is induced by immunizing experimental animals via subcutaneous injection of short myelin peptides together with specific adjuvants to boost the immune response. Like the human counterpart, EAE mice develop demyelinating lesions, immune cell infiltration into the central nervous system (CNS), glia activation and neuronal injury. A consistent body of evidence also supports a mechanistic role for B cell dysfunction in the etiology of both MS and EAE. B cells can serve as antigen-presenting cells as well as a primary source of pro-inflammatory cytokines and autoantibodies. In EAE, antibodies are generated against the myelin peptides that were employed to induce the disease. Such autoantibodies have been shown to mediate either myelin loss or pathogenic T cell reactivation into the CNS. This article describes an efficient ELISA-based protocol to quantify autoantibodies in the serum of C57BL/6J mice immunized with the myelin oligodendrocyte glycoprotein 35-55 (MOG35-55) peptide. The proposed method serves as a powerful tool to investigate the specificity and magnitude of the aberrant humoral response in the context of autoimmune demyelination.

Introduction

Multiple sclerosis (MS) is a chronic autoimmune disease of the central nervous system (CNS) characterized by focal infiltration of immune cells into the brain parenchyma, breakdown of myelin sheaths wrapping axons, glia activation, and neuronal loss¹. In addition to the well-established role of pathogenic T cells, multiple lines of evidence have highlighted the involvement of B cells in mediating the autoimmune response against the CNS. B cells undergo clonal expansion in the MS brain and antibodies against myelin components have been detected within demyelinated lesions^2,3. The selecti....

Protocol

All procedures involving mice were performed in compliance with experimental guidelines approved by the East Carolina University Institutional Animal Care and Use Committee (IACUC). Wildtype C57BL/6J female mice between 8-10 weeks of age were used in this study. The animals were obtained from a commercial source (see Table of Materials). EAE was induced following previously published reports^12,13,14.

Discussion

Here, a simple and efficient ELISA-based protocol was reported to accurately quantify the humoral response in a relevant animal model of MS pathology. This method has been recently employed to describe the novel role of the ataxin-1 protein in controlling the serum levels of autoantibodies in the MOG35-55/C57BL6J EAE paradigm¹². In this regard, a number of factors should be taken into consideration, in order to obtain consistent and biologically meaningful results with this method.

Materials

Name	Company	Catalog Number	Comments
1 mL syringes	BD Biosciences	309628
1.5 mL microcentrifuge tubes	Fisher	05-408-129
25 G needles	BD Biosciences	305122
3,3',5,5'-tetramethylbenzidine (TMB) substrate	Thermo Fisher	N301	Store at 4 °C
Adhesive seals	Thermo Fisher	AB0558
Bovine serum albumin (BSA)	Sigma	A7906	Store at 4 °C
C57BL/6J female mice	The Jackson Laboratory	000664	Animals between 8-10 weeks of age should be used for EAE experiments
Cryogenic tubes	Fisher	10-500-25
Dissection tray	Fisher	S111022
Dissector scissors	Fine Science Tools	14082-09
ELISA coating buffer	BioLegend	421701	Store at 4°C
Excel software	Microsoft		Analysis spreadsheet
Forceps	Fine Science Tools	11152-10
Goat Anti-Mouse IgG, Human ads-HRP	SouthernBiotech	1030-05	Store at 4 °C
LED light source	Fisher	AMPSILED21
Microplate reader	Fisher	14-377-575
Molecular biology grade water	Corning	46-000-Cl
Mouse MOG35-55 peptide	EZBiolab	cp7203	Store at -80 °C
Multichannel pipette	Axygen	AP-12-200-P
Noyes spring scissors	Fine Science Tools	15011-12
Nunc MaxiSorp 96-well plates	BioLegend	423501
Orbital shaker	Fisher	88-861-023
Oven	VWR	445-0024
Phosphate buffer saline (PBS)	Thermo Fisher	14190144
Refrigerated tabletop centrifuge	Thermo Fisher	75002441
Stop solution	Thermo Fisher	N600
Tween 20	Bio-Rad	1706531