Preparing an Antigen-Adjuvant Emulsion to Induce Autoimmune Encephalomyelitis

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.
   1. Add 100 mg of freeze-dried M. tuberculosis H37RA (see Table of Materials) and five 3.2 mm steel beads to a 7 mL tube (see Table of Materials). Shake the tube in a homogenizer (see Table of Materials) for 60 s at the highest speed setting (5,000 rpm).
   2. Add 5 mL of incomplete Freund's adjuvant (IFA) to the tube and shake again for 60 s at the highest speed. Transfer the slurry of homogenized M. tuberculosis in IFA (now named CFA) to a fresh tube with a pipette, leaving the beads behind, and store at 4 °C until use.
2. Add ultrapure water to the freeze-dried powder of MOG_35-55 peptide (see Table of Materials) to obtain a final peptide concentration of 10 mg/mL. Sterilize the solution by passing it through a 0.2 µm filter. Prepare 60 µL aliquots and store at -20 °C until use.
   NOTE: The MOG_35-55 peptide used for this experiment is of ImmunoGrade purity (~70%), is TFA-cleaved, dissolves easily in water, and contains a C-terminal amide (-NH₂) to increase in vivo stability. Peptide aliquots were never thawed and re-frozen more than twice, and were stored at -20 °C until use.

2. Preparation of CFA/peptide emulsions

1. Calculate the amount of emulsion needed for immunization. In this standard protocol, each mouse receives 50 µg of MOG_35-55 peptide and 300 µg of M. tuberculosis dispersed in Freund's adjuvant (CFA). The amount of each reagent (MOG_35-55 peptide, PBS, CFA, and IFA) required for preparing the emulsions can be calculated using Supplemental File 1. An additional 20% of all reagents, to compensate for the small loss of emulsion, is included in the calculation.
   NOTE: The commercial emulsion kit (see Table of Materials) used in this study comprises a tube, screw cap, and a plunger in a sterilized pouch. Each tube of the emulsion kit holds a maximum of 9.6 mL, making it possible to prepare 8 x 1 mL syringes sufficient for immunizing 40 mice (or 80 mice if using 100 µL of emulsion per animal).
2. Place the screw-capped tube (from the commercial emulsion kit) and reagents to be used on ice.
3. Add the emulsion components in the following order in the volumes calculated in step 2.1: PBS, then the peptide, then M. tuberculosis in CFA, and finally IFA.
4. Close the tube with the cap firmly by tightening and loosening it several times. Shake the tube vigorously for 5-10 s by hand to pre-mix the reagents.
5. Place the tube in the shaking homogenizer and secure it with the rod. Set the speed to the highest setting and the time to 60 s. Once the run is finished, place the tube on ice for 3 min. Repeat the run one or two more times with the same settings.
6. Centrifuge the tube at 300 x g for 1 min to remove trapped air and compact the emulsion.
7. Remove the cap from the tube, insert the plunger into the tube, and push it down slowly until it reaches the top of the emulsion. Remove the snap-off closure at the bottom of the tube by twisting it.
8. Remove the plunger from an injection syringe (1 mL; see Table of Materials). Add a needle (preferably 25-27 G) to the injection syringe.
9. Attach the back end of the injection syringe to the dedicated lock at the bottom of the tube and lock it with a short twist.
10. Transfer the emulsion from the tube to the injection syringe by pushing the plunger gently. Stop when the emulsion reaches the 0.15 mL graduation of the injection syringe.
11. Separate the injection syringe from the tube and insert the plunger carefully, taking care that no air enters the syringe. Push the plunger until the emulsion comes out of the needle.
    NOTE: At this point, some of the emulsion can be used for quality control (see step 3).
12. Repeat steps 2.8-2.11 for the rest of the emulsion present in the tube.
    NOTE: To minimize the waste of expensive CFA/antigen emulsions, 1 mL syringes should be used. Other syringes that fit the dedicated lock at the bottom of the tube may be used; however, a larger volume of emulsion may need to be prepared. The CFA/MOG_35-55 peptide emulsion can be stored at 4 °C for up to 15 weeks without losing its EAE-inducing capacity.

3. Quality control of emulsion

1. Drop-test: Add a small drop of the emulsion into a sterile 50 mL conical tube filled with 20 mL of cold water. Close the tube and shake it by hand for a couple of seconds. The appearance of tiny distinct droplets confirms the formation of a water-in-oil emulsion.
2. Examine the emulsion using phase contrast microscopy.
   1. To analyze the size of the water-in-oil particles, add a tiny drop (2-3 µL) of the emulsion to a microscope slide, smear it out with a cover slip, and then push hard in a circular motion to flatten out the emulsion.
   2. Examine the smeared emulsion under a phase contrast microscope (see Table of Materials) with 400x magnification and focus on a field with a monolayer of the emulsion. Ensure that small uniform grey/white particles are visible (Figure 2A).

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.

Figure 2: Characterization of emulsions produced using different methods. (A) Representative images from three different preparation meth...

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
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
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	Sterile filter
PBS, Ca2+/Mg2+ 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