Preparing an Antigen-Adjuvant Emulsion to Induce Autoimmune Encephalomyelitis

Experimental autoimmune encephalomyelitis, EAE, is a central nervous system disease. Autoantibodies target the myelin oligodendrocyte glycoprotein, MOG, on the myelin sheath of neurons, causing myelin destruction and nerve degeneration.

EAE induction in mice requires injecting an antigen-adjuvant emulsion of the autoantigen MOG mixed with an adjuvant — containing a bacterial antigen — to enhance the immune response.

To prepare the emulsion, take an aqueous suspension of MOG in a homogenizer tube. Place the tube on ice, preventing antigen degradation.

Add the adjuvant — inactivated pathogenic bacteria dispersed in an oil phase. Place the tube inside a shaking homogenizer. The shaking causes turbulence in the fluid, generating an emulsion.

Under a microscope, the emulsion shows uniformly dispersed, micro-sized, aqueous droplets — containing MOG — within a continuous oil phase. The homogeneity of the emulsion ensures higher reproducibility of disease induction.      

To inject the emulsion in a susceptible strain of mice, attach a syringe at the front of the tube and transfer the emulsion into it. Take an anesthetized mouse and inject the emulsion subcutaneously.

Upon injection, the oil in the emulsion ensures slow and sustained antigen release, increasing the immune response duration.

The inactivated pathogen in the emulsion causes a localized inflammatory response, recruiting immune cells to the injection site. The recruited immune cells recognize the released MOG antigen and stimulate antibody production, leading to the induction of EAE.

Place the screw-capped tube in 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 pre-mix 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 3 minutes and repeat the run 1 or 2 more times with the same settings. Centrifuge the tube at 300 x g for 1 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 end 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 the 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-in-oil particles, add a tiny drop of the emulsion to a microscope slide, smear it out with a coverslip, 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.