Polyclonal antibody preparation - Database & Sql Blog Articles

First, equipment:

Scissors (for rabbit hair), elbow ophthalmic surgery forceps (for free blood vessels), straight ophthalmic surgery scissors (for cutting blood vessels), scalpel holder, surgical blade, syringe (1ml, 10ml, 25ml) with needle, rabbit holder, sterilized flask (200ml) or plate (diameter 18cm), four elbow hemostatic forceps, two straight hemostats, surgical sutures, plastic blood vessels, gauze, etc.

Second, reagents:

Saline (or PBS), Freund’s complete adjuvant (FCA), Freund’s incomplete adjuvant (FIA), Xylene, alcohol cotton, cotton wool, 2% NaN3.

Third, immunogen:

: Protein or KLH conjugated polypeptide. Each immunization uses 100–200 μg of immunogen.

Fourth, selection of rabbits:

The rabbit should weigh more than four pounds, have smooth ears, and a healthy artery.

Fifth, immunization:

Dilute the immunogen with saline, then mix it with the corresponding adjuvant in a 1:1 ratio. Ensure the antigen and adjuvant are thoroughly mixed to form a stable emulsion, which is injected subcutaneously around the shoulders and intramuscularly into the thigh. Each area receives about 1/4 of the immunogen to ensure a sustained immune response.

Note:

Before injecting the antigen, collect some normal serum as a negative control. Once the rabbit adapts to its environment, take 5ml of blood from the ear artery after about four days. The antigen must be purified; otherwise, antibody quality will be affected. FCA or FIA must be fully emulsified before injection. Mix equal parts of antigen and adjuvant, shake vigorously on a mixer until emulsified. If not properly emulsified, the immune response may be compromised. After shaking, centrifuge at 1000rpm for one minute. If no separation occurs, the mixture is ready for injection. First immunize with FCA, then use FIA later.

Immunization method:

Use multi-point injections on the back, injecting 0.1ml per site on both sides of the spine, with 4–6 points. Inject every two weeks, selecting different sites each time to avoid ulcers. Each immunization uses about 100μg of antigen. Perform 4–5 immunizations, gradually reducing the antigen dose.

Sixth, blood collection:

One week after immunization, draw blood from the ear artery to test antibody titer. The first few immunizations produce lower titers, so test the first two samples. After three immunizations, higher titers are achieved, and up to 40ml of blood can be taken without causing anemia in the rabbit.

The first few blood samples were collected from the ear artery of a 19-week-old rabbit, and the serum was allowed to precipitate overnight at room temperature.

For the final blood collection, use the carotid artery:

1. Place the rabbit on a frame, fix the head and limbs with gauze, and slightly lower the head to expose the neck. Shave and disinfect the skin.

2. Cut along the midline of the neck with a scalpel, separate subcutaneous tissue to expose the sternocleidomastoid muscles. Be careful to avoid small blood vessels, and apply hemostasis if needed.

3. Use straight hemostatic forceps to separate the loose tissue between the sternocleidomastoid and trachea. Expose the common carotid artery, remove surrounding nerves and connective tissue.

4. Place two black silk threads under the artery, ligate the distal end, and clamp the proximal part with a vascular clamp.

5. Make a small incision in the artery between the two threads using pointed ophthalmic surgery tools. Insert a plastic tube and ligate the proximal end to secure the vessel.

6. Release the hemostat to allow blood to flow into the container. A rabbit can typically bleed 100–120ml.

Seventh, serum separation:

If blood is collected in a triangular flask or plate, place it at an angle in a 37°C oven for 2 hours, then store at 4°C overnight. The next morning, aspirate the serum with a pipette. If using a centrifuge tube, incubate at 37°C for 2 hours, then refrigerate overnight. Centrifuge at 10,000 RCF for 10 minutes. Add NaN3 to reach a final concentration of 0.02%, and store at -20°C.