A Rapid, Simple, and Standardized Homogenization Method to Prepare Antigen/Adjuvant Emulsions for Inducing Experimental Autoimmune Encephalomyelitis

Summary

To induce experimental autoimmune encephalomyelitis, an animal model of multiple sclerosis, mice are immunized with a water-in-oil emulsion containing an autoantigen and complete Freund's adjuvant. While several protocols exist for the preparation of these emulsions, a rapid, simple, and standardized homogenization protocol for emulsion preparation is presented here.

Abstract

Experimental autoimmune encephalomyelitis (EAE) shares similar immunological and clinical features with multiple sclerosis (MS), and is therefore widely used as a model to identify new drug targets for better patient treatment. MS is characterized by several different disease courses: relapsing-remitting MS (RRMS), primary progressive MS (PPMS), secondary progressive MS (SPMS), and a rare progressive-relapsing form of MS (PRMS). Although animal models do not accurately mimic all of these contrasting human disease phenotypes, there are EAE models that reflect some of the different clinical manifestations of MS. For example, myelin oligodendrocyte glycoprotein (MOG)-induced EAE in C57BL/6J mice mimics human PPMS, while myelin proteolipid protein (PLP)-induced EAE in SJL/J mice resembles RRMS. Other autoantigens, such as myelin basic protein (MBP), and a number of different mouse strains are also used to study EAE. To induce disease in these autoantigen-immunization EAE models, a water-in-oil emulsion is prepared and injected subcutaneously. The majority of EAE models also require an injection of pertussis toxin for the disease to develop. For consistent and reproducible EAE induction, a detailed protocol to prepare the reagents to produce antigen/adjuvant emulsions is necessary. The method described here takes advantage of a standardized method to generate water-in-oil emulsions. It is simple and fast and uses a shaking homogenizer instead of syringes to prepare quality-controlled emulsions.

Introduction

A breakdown of immunological tolerance can result in the generation of autoimmune disorders, such as multiple sclerosis (MS). It is estimated that 2.8 million people are living with MS worldwide¹. Although the exact cause of MS is still largely unknown, dysregulation of autoreactive T and B cells, as well as defects in Treg function, play important roles in the pathogenesis of the disease^2,3.

Animal models of autoimmune diseases are essential tools to investigate potential therapeutic modalities. The experimental autoimmune encephalomyelitis (EAE) model has b....

Protocol

All animal procedures were performed according to the practices of the Swedish Board of Animal Research and were approved by the Animal Ethics Committee, Lund-Malmö, Sweden (Permit number: M126-16).

NOTE: A schematic flow of the method is described in Figure 1.

1. Material preparation

NOTE: Prepare all the reagents aseptically in a sterile hood, and aliquot and store at the indicated temperature. The reagents can be stored up to 2 years without losing their effect.

1. Prepare CFA containing 20 mg/mL Mycobacterium tub....

Results

The rapid, simple, and standardized protocol for the preparation of CFA/MOG emulsions is depicted in Figure 1. This method has recently been described elsewhere⁶. The CFA/MOG emulsions can also be prepared with other methods, such as the traditional syringe method or by vortexing. These methods were compared here by assessing the quality of the emulsions. All the methods produced water-in-oil emulsions; the homogeneity and quality of these emulsions were assessed by a.......

Discussion

Water-in-oil emulsions, such as antigen/Freund's adjuvant, have been used for more than half a century to induce EAE¹⁷. There is currently no standardized method to prepare antigen emulsions that is independent of human influence. Manual mixing using syringes is standard for most laboratories, however this method is time consuming, often results in an excessive loss of material, and the quality differs depending on the scientist preparing it.

The method presented in.......

Materials

Name	Company	Catalog Number	Comments
1 mL Injection syringe	B. Braun	9166017V
1 mL Injection syringe	Sigma-Aldrich	Z683531
7 ml empty tubes with caps	Bertin-Instruments	P000944LYSK0A.0	7 mL tube
50 mL sterile centrifuge tube	Fisher Scientific	10788561	50 mL tube
Bordetella pertussis toxin	Sigma-Aldrich	P2980	Store at -20 °C
Dispersant, light mineral oil	Sigma-Aldrich	M8410	Store at RT
Emulsion kit	Bertin-Instruments	D34200.10 ea	Containing a tube, cap, and plunger
Incomplete Freund's Adjuvant	Sigma-Aldrich	F5506	Store at +4 °C
Mycobacterium tuberculosis, H37RA	Fisher Scientific	DF3114-33-8	Store at +4 °C
Mastersizer 2000	Malvern Panalytical	N/A	Particle size analyzer
Minilys-Personal homogenizer	Bertin-Instruments	P000673-MLYS0-A	Shaking homogenizer
MOG 35-55 Peptide	Innovagen	N/A
Montanide ISA 51 VG	Seppic	36362Z	FDA-approved oil adjuvant
Pall Acrodisc Syringe Filters 0.2 μm	Fisher Scientific	17124381	Sterlie filter
PBS, Ca2+/Mg2+ free	Thermo Fisher Scientific	14190144	PBS
Phase-Constrast Microscope	Olympus	BX40-B
Steel Beads 3.2 mm	Fisher Scientific	NC0445832	Autoclave and store at RT
Triton X-100	Sigma-Aldrich	648463	Store at RT