Experimental reagent saline (or PBS)
Freund's complete adjuvant (FCA)
Freund's incomplete adjuvant (FIA)
Xylene, alcohol cotton, absorbent cotton, 2% NaN3
Laboratory equipment Scissors (for rabbit hair cutting), elbow eye surgery forceps (for free blood vessels), straight eye surgery scissors (for blood vessel cutting), scalpel holder, surgical blade, syringe (1ml, 10ml) , 25ml) with needle, rabbit holder, sterilized Erlenmeyer flask (200ml) or plate (diameter 18cm), four elbow hemostatic forceps, two straight hemostatic forceps, surgical sutures, plastic blood vessels, gauze, etc.
Experimental materials 1. Immunogen: protein or KLH coupled peptide. 100-200μg immunogen per immunization.
2. The choice of rabbit, the weight of the rabbit should be more than four pounds, both ears are smooth, the ear stills, arteries and health are obviously visible.
Experimental procedure 1. Immunization
The immunogen was diluted with normal saline and then mixed 1: 1 with the corresponding adjuvant. The antigen and adjuvant are completely mixed to form a stable emulsion, which is injected subcutaneously under the skin around the shoulders of the rabbit and intramuscularly in the posterior thigh. Approximately 1/4 of the immunogen is used in each area. In this way, the immunogen can persist to improve the immune response.
Note:
Before the antigen injection, some normal serum needs to be collected and used as a negative control when the antibody has been prepared. After the rabbit is stable in the new environment, it takes about four days to collect blood from the ear artery. About 5ml of blood is enough.
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 with the adjuvant in equal proportions, it is placed on a mixer and shaken vigorously to fully emulsify the antigen. The emulsification process is time-consuming and laborious. However, if the emulsification is insufficient, it will affect the immune effect. After shaking, centrifuge at 1000 rpm Minutes, such as water and oil phase can be injected without layering. FCA was used for the first immunization, and FIA was used in the future.
The method of immunization can use the back multi-point injection method. That is, choose 4-6 points of subcutaneous injection on both sides of the rabbit's spine, and inject 0.1ml per point, and choose different points of injection in the above sites after 2 weeks interval (don't choose the adjacent site, otherwise the ulcer will not heal well). The amount of antigen per immunization is about 100 μg. The number of immunizations can be four or five times, and the large amount of antigen can reduce the number of immunizations.
2. Blood sampling
After one week of immunization, blood of the ear artery can be used to detect antibody titer. Because the titers of antibodies produced in the first few immunizations are relatively low, the first two blood draws are sufficient for testing. After three immunizations, you can obtain a more efficient antibody. The amount of blood taken each time can be 40ml. Do not take too much, otherwise it will cause rabbit anemia.
The first few blood samples were taken from the rabbit's ear artery with a 19-gauge needle, and serum was precipitated at room temperature overnight.
Carotid artery bleeding can be used for the last blood collection. The specific operations are as follows:
1) The rabbit lies on its back on a rabbit stand to fix its head, and use gauze to fix its limbs. Lower your head slightly to expose your neck. Shave and disinfect the skin.
2) Cut the skin approximately 10 cm along the midline of the neck with a scalpel, and separate the subcutaneous connective tissue until the sternocleidomastoid muscles on both sides of the trachea are exposed (be careful, if hemorrhage from small blood vessels is encountered, hemostatic forceps can be used to stop the bleeding).
3) Use straight-head hemostatic forceps to separate the loose tissue of the cervical triangle between the sternocleidomastoid muscle and the trachea. After the common carotid artery is exposed, it is freed with curved ophthalmic forceps and the nerve and connective tissue are stripped.
4) Insert two black silk threads under the artery and place them at the telecentric and proximal ends. Ligate the wire at the telecentric end. The arteries at the proximal end are clamped with vascular clamps.
5) Use a small thumb pad under the blood vessel, and use a pointed ophthalmic surgical scissors to cut a small mouth (do not cut) on the arterial wall between the two silk threads and insert the plastic blood vessel. Then ligature and fix the silk thread at the proximal end to the blood vessel to prevent the blood vessel from slipping off.
6) Loosen the hemostatic forceps and allow blood to flow into the container. Generally a rabbit can bleed 100-120ml.
3. Separation of serum
If a flask or plate is used to hold the blood, place the vessel in a 37oC oven for 2 hours at an angle, transfer to 4oC for precipitation overnight, and use a pipette to aspirate the serum the next morning. If collected with a centrifuge tube, place it in a 37oC oven for 2hr, transfer to 4oC for precipitation overnight, and centrifuge the next morning at 10,000 RCF for 10 minutes. Add NaN3 to the serum to a final concentration of 0.02% and store at -20oC after aliquoting.
Woven Greenhouse Film,Tunnel Greenhouse Film,Greenhouse Uv Plastic Film,Clear Woven Greenhouse Film 12 Mil
Henan Fengcheng Plastic Co.,ltd , https://www.fcsyplastic.com