Preparing an Antigen-Adjuvant Emulsion to Induce Autoimmune Encephalomyelitis

Abstract

Source: Bäckström, B. T. A Rapid, Simple, and Standardized Homogenization Method to Prepare Antigen/Adjuvant Emulsions for Inducing Experimental Autoimmune Encephalomyelitis. J. Vis. Exp. (2022)

This video demonstrates an efficient homogenization technique to generate an antigen-adjuvant emulsion to induce experimental autoimmune encephalomyelitis (EAE) in a murine model. The emulsion contains an autoantigen — myelin oligodendrocyte glycoprotein (MOG) — in the water phase, surrounded by a continuous oil phase comprising an inactivated pathogenic bacteria. The emulsion ensures a sustained release of antigens for a prolonged immune response.

Protocol

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.

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 for up to 2 years without losing their effect.

1. Prepare CFA containing 20 mg/mL Mycobacterium tuberculosis as described below.

Results

Figure 1: Schematic of the process of emulsion preparation using a commercial emulsion kit. This figure has been reused from Topping et al with permission.

Materials

List of materials used in this article

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
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
<em>Mycobacterium tuberculosis</em>, H37RA	Fisher Scientific	DF3114-33-8	Store at +4 &deg;C
Minilys-Personal homogenizer	Bertin-Instruments	P000673-MLYS0-A	Shaking homogenizer
MOG<sub>35-55</sub> Peptide	Innovagen	N/A
Montanide ISA 51 VG	Seppic	36362Z	FDA-approved oil adjuvant
Pall Acrodisc Syringe Filters 0.2 &mu;m	Fisher Scientific	17124381	Sterile filter
PBS, Ca<sup>2+</sup>/Mg<sup>2+</sup> free	Thermo Fisher Scientific	14190144	PBS
Phase-Contrast Microscope	Olympus	BX40-B
Steel Beads 3.2 mm	Fisher Scientific	NC0445832	Autoclave and store at RT