Protocol for the Preparation of Anti-Red Blood Cell Antibodies (Hemolysin)

An antibody is an immune-specific globulin produced by the body after stimulation by an antigen. In immunological practice, to prepare antibodies, antigenic substances (bacteria, viruses, toxoids, serum, and other proteins) are often injected into animals. After a certain period, a large number of specific antibodies can be produced in the animal's serum. This serum containing specific antibodies is called immune serum. Immune serum is not only important for the diagnosis, prevention, and treatment of infectious diseases, but also important for organ transplantation, tumors, and certain scientific research.

Preparation of red blood cell suspension

• The anticoagulated blood is first precipitated by centrifugation (2000 cpm x 5 minutes) and the supernatant is aspirated. Add 2-3 times saline and mix by repeated blows with a capillary burette, centrifuge for 5 minutes, and aspirate the supernatant. Wash 3 times in a row, the last time can be centrifuged for 10 minutes to make the blood cells dense at the bottom of the tube. Until the supernatant is clear and colorless, the supernatant should be aspirated again, and the blood cells should be kept in a dense mass.
• Use saline to prepare the red blood cells to the required concentration, i.e., the blood cell suspension for the test. If 10% suspension is required, 1 ml of washed red blood cells and 9 ml of saline should be added.

Immunization methods

• Select 2-3 kg healthy male rabbits and immunize them according to the following dose and procedure. Day 1 whole blood injection 0.5 intradermal, day 3 whole blood injection 1.0 intradermal, day 5 whole blood injection 1.5 intradermal, day 7 whole blood injection 2.0 intradermal, day 9 whole blood injection 2.5 intradermal, day 12 20% red blood cell suspension whole blood injection 1.0 IV, day 15 20% red blood cell suspension whole blood 1.0 IV.
• 7 days after the last injection, ear vein blood collection 1 ml. Titrate the blood unit up to 1:2000 or more can be carotid artery bloodletting, separation of serum. Put an equal amount of glycerol antiseptic, divide it into sterile ampoules, and store it at 4°C for backup.

Sheep blood taken and mixed with sterilized preservation solution in equal parts and placed in the refrigerator can be stored for several weeks, or anticoagulation methods can be used.

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