Amino Acids, Peptides and Proteins - Dévényi, T., Gergely, J. 1976

Methods of Immunochemical Analysis
Preparation of Immune Serum
Immunization Protocols with Adjuvants

A. Adsorption of antigen on aluminum hydroxide: adsorption of a protein antigen on aluminum hydroxide gel significantly increases its immunogenicity.

In our laboratory, aluminum hydroxide is used during the immunization of rabbits to obtain antiserum against human Serum Proteins (e.g., for immunoelectrophoretic studies). The method for preparing the antiserum is described below.

Antigen preparation consists of mixing the following components: 6.25 ml of human serum (a pool of sera from several Donors is used), 12.5 ml of 0.9% NaCl solution, 5.0 ml of aluminum hydroxide gel, and 1.25 ml of 5% phenol solution. 2.5 ml of this mixture is injected intramuscularly into the animals at two sites once a week for 6 weeks. One week after the final injection, Blood is drawn from the rabbit to determine the antibody titer. If the serum titer has not yet reached the required level, or if the desired number of antigenic components cannot be detected, weekly injections are repeated until the desired effect is achieved.

B. Freund's adjuvant [7] is of significant assistance in obtaining antiserum. The required amount of protein antigen (e.g., 500 mg of Ovalbumin) is dissolved in 10 ml of physiological saline containing 1% phenol. To the antigen solution, 10 ml of a commercially available lanolin-derivative emulsifier (Aquaphor from Duke Lab., Falba from Pfaltz and Bauer Inc. N.Y., or anhydrous lanolin) is added, and mixed in a Waring blender. To the resulting homogenate, 20 ml of liquid paraffin containing 50 mg of uniformly suspended, heat-killed lyophilized Mycobacterium tuberculosis is added. After re-homogenization, 1 ml of the resulting mixture, warmed to 37°C before injection, is administered to the animals intramuscularly or subcutaneously three times at weekly intervals.

Commercially prepared complete and incomplete Freund's adjuvants are available. The following immunization schedule for protein Antigens has proven highly effective. 0.1–1.0 mg of antigen is dissolved in 0.25–0.5 ml of physiological saline and homogenized with an equal volume of complete Freund's adjuvant. The emulsion is injected intramuscularly at four sites. After 3–4 weeks, blood is drawn from the animal to determine the antiserum activity by Immunoelectrophoresis. If necessary, this Procedure is repeated. If, 2–3 weeks after booster immunization, the specific activity of the antiserum remains insufficient, additional weekly intramuscular injections of 5–10 mg of protein antigen without adjuvant are given until the antibody titer reaches the desired level.

NOTES

1. Preparation of aluminum hydroxide gel. Solutions: a) 11.0 g of ammonium sulfate is dissolved in distilled Water, the final volume of the solution is adjusted to 300 ml, and it is heated to 63°C;

b) 38.35 g of potassium alum is dissolved in 500 ml of distilled water, and the solution is heated to 58°C.

The ammonium sulfate solution is mixed with the alum solution (1:2), the pH of the resulting mixture is adjusted to 6.5 using NaOH, then heated to 61°C and kept at this Temperature for 10 min. As soon as the formation and settling of the precipitate is complete, the mixture is centrifuged, and the precipitate is washed three times with distilled water to which a few drops of ammonia have been added. The washed precipitate is suspended in distilled water to the desired concentration.

2. Once the serum activity, monitored by taking test samples during immunization, reaches the desired level, the producer animals are bled to obtain the maximum volume of serum. If the immune serum is obtained under non-sterile conditions, it is recommended to add merthiolate at a ratio of 1:5000. When stored in a refrigerator (preferably frozen), The activity of the obtained antisera can be preserved for several years.

If the immunization course of the producer animal does not end with total bleeding, a booster injection of the antigen can be given after some time at the same dose and by the same route; 7–9 days later, blood can be drawn to obtain the immune serum.

3. Immune serum used in precipitation reactions should have an antibody titer of at least 1:10 000.