The quality of the emulsion for immunization is critical for achieving robust antibody titers against an antigen in immunized mice, especially developing collagen-induced arthritis with high incidence and severity. Although emulsions can be made using various methods, Chondrex, Inc. strongly recommends a homogenizer method. Alternative methods, double-syringe methods or sonication methods often yield emulsions with inconsistent quality and are unstable for effective immunization. Moreover, sonication carries the additional risk of antigen fragmentation.
To ensure consistent and effective immunization, Chondrex, Inc offers the following homogenizer-based emulsion preparation protocol for your successful immunization in mice.
Video Tutorial - Click the image
Protocol
A.Materials
1. Homogenizer- An electric homogenizer is highly recommended for preparing an emulsion (Figure 1)
2. A homogenizer blade (a), a syringe with a 3-way stopcock, and a hamilton glass syringe (c) (Figure 2)
B. Procedures
1. Use a homogenizer with a small blade (diameter of 5 mm or less) to emulsify an adjuvant and an antigen solution. Seal the tip of the syringe with a 3-way stopcock. Next, clamp the syringe to a ring stand and place it in an ice water bath to keep the emulsion cool during mixing, as heat may denature your antigens (Figure 3).
2. Add one volume (maximum: 25% of the syringe volume) of the adjuvant to the end of the syringe sealed with the 3-way stopcock. Then gradually add an equal volume of the antigen solution dropwise while mixing at low speed (1000-3000 rpm). Typically use 2 mg/ml antigen solution for 100 ug/mouse immunization.
NOTE: To ensure a high-quality emulsion, the maximum emulsion volume should be no more than half of the syringe volume (50%). If more is needed, make several batches.
3. Continue mixing the emulsion at maximum speed (approximately 10,000 - 30,000 rpm) for 2 minutes. Cool down the emulsion by keeping the syringe in the ice water bath for 5 minutes. Repeat mixing and cooling 2-3 times. For
larger volumes (2-5 ml), we suggest moving the blade throughout the emulsion while mixing for better uniformity.
4. Replace the stopcock with a needle and test the stability of the emulsion by adding one drop of emulsion into a beaker of water. If the emulsion is stable, the drop will remain as a solid clump which does not dissipate on the water’s surface. If the emulsion dissipates onto the water surface, then the emulsion is not stable. Add a few drops of adjuvant, mix again, and retest.
5. Transfer the emulsion to a 1 ml Hamilton glass syringe (Figure 2c). Injecting an accurate volume of emulsion is
difficult with a plastic syringe.
NOTE 1: Remove air bubbles from the emulsion by forcefully swinging your arm towards the floor, with the Hamilton
syringe in hand (plunger side down).
NOTE 2: Chondrex, Inc. recommends injecting the collagen emulsion within an hour of preparation. Keep the emulsion at 4°C until use.