Protein Protocols Protein Protocols

1110 Page and Thorpe
final quantity will be 5–50 µg, since the preparation will be diluted with an equal volume
of adjuvant). Mix the immunogen solution with an equal volume of FCA and draw up into
a glass syringe or prepare directly in the syringe barrel.
2. Remove excess air from the syringe barrel, and connect to a second glass syringe with its
plunger fully depressed via a double-hub connector.
3. Ensure the connections are tight and not leaking, and transfer the oil and immunogen
solutions from one syringe to the other. Continue this action until the mixture is fully
emulsified when it should appear as a creamy, white thick liquid.
4. Transfer emulsion into one of the glass syringes, remove from double-hub connector and
empty syringe, and fit a small-diameter needle (the size of which will depend on the animal
to be immunized and route of immunization).
3.2. Immunization Procedure
1. Prime mice or rats by immunizing with immunogen subcutaneously on the flanks and
neck (0.1 mL/site, 3–5 sites). Do his by raising the skin between thumb and forefinger,
and inserting the needle into the raised area at a shallow angle. A short narrow-diameter
needle is preferred (0.4 × 27 mm) to avoid injection into the deeper body layers/cavities.
The result should be a discrete lump under the skin.
2. Boost intraperitoneally after 14–28 d using the immunogen prepared in PBS via a short
narrow-diameter needle. Administer the immunogen at one site in no more than 0.5 mL
using the same total dose as that used for priming (usually 5–50 µg).
3. Three days after the booster immunization, withdraw blood from the tail vein with a needle
(0.4 mm) and syringe, and use this as a positive control in screening assays during hybridoma
production and as a check on the success of the immunization. Sacrifice mouse, and
remove spleen aseptically.
4. Notes
1. Shake the complete adjuvant before use to disperse the Mycobacterium particles fully.
2. The emulsion is very difficult to recover completely from the walls of vessels, and so
forth; therefore, it is inevitable that some will be lost during preparation. To minimize
this, prepare the emulsion in glass syringes, one of which can be used for the immunization.
If possible, prepare slightly more than required to compensate for losses, which normally
amount to around 10%.
3. If Luer connectors are not available, the emulsion can be prepared by vigorous shaking or
mixing using a whirlimixer. This is less efficient at producing an emulsion and requires
larger volumes of immunogen.
4. When using connected syringes, keep the hub connector as short as possible to avoid
emulsion loss.
5. Use glass syringes to prepare the emulsion since the rubber seals of plastic syringes are
not compatible with the mineral oil of the adjuvant.
Reference
1. Thorpe, R. (1994) Producing antibodies, in Immunochemistry Labfax (Kerr, M. A. and
Thorpe, R., eds.), Bios Scientific, Oxford, UK, pp. 63–81.