Protocols for Preparation and Potency Determination of Immune Serums

The traditional method of preparing immune serum is to inject the antigen into the animal body, and the B cells in the animal body will proliferate and differentiate into plasma cells to produce antibodies. There are many methods to determine the antibody potency in immune serum, such as agglutination test, precipitation test, hemolysis test, ELISA, etc. All of them can be used for the determination of antibody potency, and the hemolysis test will be used in the present experiment to determine the potency of the obtained immune serum.

• The first immunized mice are injected intraperitoneally with 0.2 ml of 20% SRBC. 4 days later, the mice are reinforced with the same dose and the same route.
• 7 days after the initial immunization, blood is collected from the eyeballs and placed in a small test tube with an anticoagulant. 2,000 rpm centrifugation is performed for 1 minute, and the supernatant is the immunized plasma.
• The mouse anti-SRBC immunized plasma obtained earlier is first diluted 10-fold to make 1:10 immunized plasma.
• Three tubes numbered A, B, and C, are used for 3-, 4-, and 5-fold dilutions of 1:10 immune plasma, i.e., 1:30 immune plasma in tube A, 1:40 immune plasma in tube B, and 1:50 immune plasma in tube C. The plasma is diluted 10-fold in tube A, and 1:40 in tube C. The plasma is then centrifuged at 2000 rpm for 1 minute.
• Take 15 test tubes and divide them into three columns, A, B, and C, with 5 tubes in each column. First, 0.5 ml of saline is added to each tube, then 0.5 ml of plasma is aspirated from tubes A, B, and C and added to the first tube of the corresponding columns, and then the columns are subjected to multiple dilutions.
• Add 0.25 ml of SRBC and 0.25 ml of complement to each tube.
• Mix the liquid in each tube and place in a 37°C water bath for 30-40 minutes.
• The highest serum dilution tube in which complete hemolysis occurred is used as the potency determination tube, and its serum dilution is the potency of the immune serum.

• When immunizing animals, take care to ensure that the SRBC is injected into the abdominal cavity, and avoid injecting into other organs (e.g., intestines, bladder).
• Try to be as precise as possible when diluting the serum and be careful not to mix pipettes used for different columns.

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