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

Experimental autoimmune encephalomyelitis or EAE is an animal model used to develop new and improved treatments for multiple sclerosis patient. Inducing disease in EAE requires a perfect oil and water emulsion to achieve consistent results. The method of emulsion preparation presented here is free from human influence, thereby limiting inconsistencies from one experiment to another.

Even though the method has been optimized for EAE, it can also be used for other autoimmune disease model and also to test cancer vaccines. To prepare the CFA slurry at 100 milligrams of freeze-dried M tuberculosis H37 RA and five, 3.2 millimeters steel beads to a seven milliliter tube. Shake the tube in the homogenizer for 60 seconds at the highest speed setting.

Add five milliliters of IFA adjuvant to the tube and shake again for 60 seconds at the highest speed. Then transfer the slurry to a fresh tube with a pipette leaving the beads behind and store at four degrees Celsius until use. To prepare a CFA/Peptide emulsion, download supplemental file one to calculate the amount needed for each reagent.

Then place the screw capped tube and reagents to be used on ice. Next, add the emulsion components. First PBS, then the peptide, the CFA slurry.

And finally, IFA. Close the tube with the cap firmly by tightening and loosening it several times. Shake the tube vigorously for 5 to 10 seconds by hand to premix the reagents.

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 seconds. Once the run is finished, place the tube on ice for three minutes and repeat the run one or two more times with the same settings.

Centrifuge the tube at 300 G from one minute to remove trapped air and compact the emulsion. 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 enclosure at the bottom of the tube by twisting it. Next, remove the plunger from all injection syringes and add a needle to the injection syringes. Then attach the back of the injection syringe to the dedicated lock at the bottom of the tube and lock it with a short twist.

Transfer the emulsion from the tube to the injection syringe by pushing the plunger gently. Stop when the emulsion reaches the 0.15 milliliter graduation of the injection syringe. Separate the injection syringe from the tube and insert the plunger carefully taking care that no air enters this syringe.

Push the plunger until the emulsion comes out of the needle and repeat the process for the rest of the emulsion present in the tube. To analyze the size of the water and oil particles add a tiny drop 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. Examine the smeared emulsion under a phase contrast microscope with 400 times magnification and focus on a field with a monolayer of the emulsion.

Ensure that small, uniform, gray or white particles are visible. Emulsions produced by the homogenization method displayed gray or white bean shaped particles representing tiny droplets of water and oil emulsions. In contrast, the emulsion droplets produced using the syringe method were larger and wider.

In the vortex method, large gray or black particles were observed corresponding to air bubbles trapped in the emulsion. Changes in particle size distribution over time measured using a laser defraction particle size analyzer for water and oil emulsions did not show any change in particle size over a period of six weeks. The fresh 3 week old and 15 week old emulsions all induced similar EAE diseases in all animals tested throughout the experiment.

Mice developed a similar disease pattern in all experiments revealing that the homogenization method presented here produced emulsions that induced EAE in a consistent and reproducible manner. Before making the emulsion for an experiment, download supplemental file number one to calculate the amount of each component needed to minimize waste. The method presented here could be used to standardize emulsion preparation not only for preclinical research, but also in the future to prepare cancer vaccines in the clinic.