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

Immunochemical Analysis Methods
Preparation of Immune Serum
Immunization protocols without adjuvant

A. Daily During the first five days, increasing amounts of a protein antigen solution are injected into the rabbit's ear vein at doses of 1, 2, 4, 8, and 16 mg (the serum protein concentration is typically around 7%). This is followed by a one-week interval, after which the series of injections is repeated. During the fourth week of immunization, the animal is injected subcutaneously with an antigen solution containing 25 mg of protein; over the next 3 days, an additional 20 mg of protein is injected daily into the ear vein. On days 7–9 after the final injection, Blood is collected from the animal, serum is harvested, and the antibody titer is determined [11].

B. The immunization course lasts 4 weeks. A single dose consists of 1–2 ml of a 1% protein antigen solution. During the first week, a single injection is given into the ear vein daily for the first three days. In each of the subsequent three weeks, immunization is performed intraperitoneally on the first day, and intravenously on the second and third days. One week after the immunization course, blood is collected, serum is harvested, and the antibody titer is determined.

C. To obtain rabbit antiserum against human Serum Proteins, the immunization method described below is recommended, as it apparently yields the best immune serum for detecting numerous protein components, including IMMUNOGLOBULINS. This method combines two immunization schedules: Milgrom's and Proom's.

First, rabbits are immunized according to Milgrom's method. To do this, 2 ml of blood is taken from the animal's ear vein, mixed with 0.5 ml of a 3.8% sodium citrate solution, the erythrocytes are precipitated and washed three times with five volumes of physiological sodium chloride solution. To the pellet of washed erythrocytes, two volumes of inactivated human group AB serum are added, and, with periodic stirring, the mixture is incubated for 1 h at room Temperature. Then the erythrocytes are washed again three times, the suspension volume is adjusted to 2 ml with saline, and injected intravenously into the same rabbit. These injections are repeated every 3 days until the antiglobulin antibody titer reaches 1: 1000—2000 in a passive hemagglutination reaction with Rh (D)+ erythrocytes sensitized with incomplete anti-D Antibodies with a titer of 1:32.

As soon as the antiglobulin antibody titer reaches the specified value, immunization is continued according to Proom's schedule. For this, human serum proteins are adsorbed onto potassium alum; 25 ml of sterile serum is diluted with 80 ml of distilled Water, and 90 ml of a 10% potassium alum solution is added to the resulting mixture. The pH of the solution is adjusted to 6.5 using 5 N NaOH. The resulting precipitate is collected by centrifugation, the supernatant is discarded, and the pellet is washed twice with 200 ml of saline containing merthiolate (1:10 000). The final volume of the suspension is adjusted to 100 ml with saline and stored at +4°C for no more than 14 days. Animals are injected intramuscularly in the thigh area with 5 ml of this suspension 2–3 times at 14-day intervals. On days 9–10 after the final immunization, blood is collected from the animals. The immunization process can be monitored immunoelectrophoretically by analyzing samples collected at the time of each subsequent antigen injection.