First, equipment
Scissors (for rabbit hair), elbow ophthalmic surgery forceps (for free blood vessels), straight ophthalmic surgery scissors (for blood vessel cutting), scalpel holder, surgical blade, syringe (1ml, 10ml, 25ml) ) with needle, rabbit holder, sterilized flask (200ml) or plate (18cm in diameter), four elbow hemostatic forceps, two straight hemostats, surgical sutures, plastic blood vessels, gauze, etc.
Second, the reagent
Saline (or PBS)
Freund's complete adjuvant (FCA)
Freund's incomplete adjuvant (FIA)
Xylene, alcohol cotton, cotton wool, 2% NaN3
3. Immunogen: a protein or KLH coupled polypeptide. 100-200 μg of immunogen is immunized each time.
Fourth, the choice of rabbit
The weight of the rabbit should be more than four pounds, the ears are smooth, and the ear is quiet, the artery is healthy.
Five, immunity
The immunogen was diluted with physiological saline and then mixed 1:1 with the corresponding adjuvant. The antigen and adjuvant are thoroughly mixed to form a stable emulsion which is subcutaneously injected under the skin around the shoulders of the rabbit and intramuscularly injected into the thigh. Approximately 1/4 of the immunogen is used in each region. Thus the immunogen can persist for an immune response.
Note:
Some normal serum needs to be collected before the antigen injection, and it is used as a negative control when the antibody is prepared. When the rabbit is stable in the new environment, it takes about four days to take blood from the ear artery. The blood volume is about 5 ml.
The antigen used for immunization must be purified, otherwise it will affect the quality of the antibody. The antigen can only be injected after being fully emulsified by FCA or FIA. After mixing the antigen solution and the adjuvant in equal proportions, placing it on the mixer to make it violently oscillate to fully emulsify the antigen, the emulsification process is time consuming and laborious, but if the emulsification is insufficient, the effect of the immunity is affected, and after shaking, the centrifuge is centrifuged at 1000 rpm. In minutes, the water phase and the oil phase can be injected without delamination. FCA was used for the first immunization and FIA was used later.
The immunization method can be performed by multi-point injection on the back. That is, choose 4-6 points of subcutaneous injection on both sides of the rabbit's spine, 0.1 ml per point. After 2 weeks interval, select different points in the above-mentioned parts (do not choose adjacent sites, otherwise the ulcers will not heal well). The amount of antigen per immunization was about 100 μg. The number of immunizations can be four or five times, and the amount of antigen can be reduced by a large number.
Sixth, take blood:
After one week of immunization, blood can be taken from the ear artery to detect antibody titer. Because the antibody produced by the first few immunizations has a lower titer, the first two blood samples are taken for testing. After three immunizations, higher titers of antibodies can be obtained. The amount of blood taken can be 40 ml each time. Do not take too much, otherwise it will cause anemia in rabbits.
The first few blood samples were taken from the rabbit's ear artery with a 19 gauge needle, and serum was precipitated overnight at room temperature.
The last time you take the blood, you can use the carotid artery to bleed. The specific operation is as follows:
1. The rabbit is placed on the rabbit frame and fixed on the head, and the limbs are fixed with gauze. The head is slightly lowered to expose the neck. Shave and disinfect the skin.
2, cut the skin about 10cm with a scalpel along the midline of the neck, and separate the subcutaneous connective tissue until the sternocleidomastoid muscles on both sides of the trachea are exposed (careful operation, such as hemorrhage caused by small blood vessels, hemostasis can be used to stop bleeding).
3. Use a straight hemostat to separate the loose tissue of the cervical triangle between the sternocleidomastoid and the trachea. After exposing the common carotid artery, use the elbow ophthalmology to dissociate it, and peel off the nerve and connective tissue.
4, under the artery into the two black silk thread, placed in the telecentric and near the heart end. Ligation of the silk thread at the far end. The arteries near the heart are clamped with a blood vessel clamp.
5, with a small thumb pad under the blood vessels, with a pointed ophthalmic surgery cut a small mouth (not cut) on the arteries between the two wires, inserted into the plastic blood vessels. The proximal end of the wire is then ligated and fixed on the blood vessel to prevent the blood vessel from slipping off.
6. Loosen the hemostat and let the blood flow into the container. Generally, a rabbit can bleed 100-120ml.
Seven, serum separation
If the blood is filled with a triangular flask or a plate, place the vessel in a 37oC oven for 2 hr, transfer to 4oC for precipitation overnight, and pipette the serum the next morning. If collected with a centrifuge tube, place in a 37oC oven for 2 hr, transfer to 4oC for precipitation overnight, and centrifuge the next morning for 10,000 RCF for 10 minutes. NaN3 was added to the serum to a final concentration of 0.02%, and stored at -20oC after dispensing.
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