Experiments on the preparation of immune serum
Experiments on the preparation of immune serum
The preparation of immune serum is a commonly used immunological laboratory technique. Highly efficient and specific immune serum can be used as a reagent for immunological diagnosis (e.g., for the preparation of immunolabeled antibodies, etc.) and for specific immunotherapy. (Source: Basic Medical Immunology Laboratory Guide, edited by Jin Boquan and Li Enshan, 1st edition, Beijing: World Book Publishing House, 1990).
Operation method
Experiments on the preparation of immune serum
Principle
Immunogenic antigens can stimulate the corresponding B cells to proliferate and differentiate to form plasma cells and secrete specific antibodies. Since different decision clusters on the surface of the antigen molecule are recognized by B cell clones with different specificities, the antibodies produced after stimulation of a certain antigen are in fact a mixture of antibodies directed against different decision clusters on the surface of the antigen molecule (i.e. polyclonal antibodies). In addition, the production of antibodies is characterized by the regularity of the memory response, which is now very different between the antibody response after the initial immunization and after the re-immunization. This is due to the involvement of memory B cells in the re-response.
Materials and Instruments
Rabbit Move I. Methods of immunization Caveat 1. Antibody reactivity of laboratory animals used for immunization varies greatly among individuals, so at least two or more animals should be used for immunization. In addition, pregnant animals should not be used. 2. The antigen for immunization must be fully emulsified by FCA or FIA before injection, otherwise the immunization effect of the antigen will be significantly affected. The above method of preparing emulsified antigen is time-consuming and laborious. It is prepared by the H-81 micro-oscillating mixer method or the three-lumen tube milling method. The former is to mix the antigen liquid and adjuvant proportionally, and then put it on the mixer to make it oscillate violently (frequency 2900 rpm, amplitude 6 mm); the latter is to mix the adjuvant and antigen liquid for immunization proportionally, and then inhale it into a syringe, and then connect the syringe with a three-lumen tube to repeatedly suction and grind it. Both methods can fully emulsify the antigen in about 1 hour. 3. On the one hand, the adjuvant can improve the effect of specific immune response to obtain highly effective immune serum, but if the antigen is impure, it can cause very small amounts of contaminants (0.005 mgN) in the antigen to produce antibodies, thus causing the purity of the immune serum to be affected. In addition, some strains of laboratory animals are allergic to BCG, especially guinea pigs and to a lesser extent rabbits, which can cause a metamorphic reaction and lead to immunization failure when the complete adjuvant is injected again. For this reason, the content of BCG in the adjuvant should be reduced or changed to incomplete adjuvant during the second immunization injection to reduce and prevent the occurrence of metamorphic reaction. 4. The dose of antigen is determined by the type of antigen. The dose of highly immunogenic antigen should be relatively small, while the dose of weakly immunogenic antigen can be relatively large. The dosage of antigen is usually calculated on a body weight basis. With the use of adjuvants, a total dose of 0.5 mg/kg body weight is appropriate for a single injection. The dose can be increased by a factor of 10 if no adjuvant is added. In addition, a small number of injections may be given for a long immunization period, and a larger number of injections may be given for a short immunization period. 5. The method of immunization can also be used to immunize the animals with multi-point injection. That is, in the rabbit spinal column on both sides of the selection of 4-6 points of subcutaneous injection, at the same time on both sides of the shoulder (or arm) and groin each injection. Each point is injected with 0.2 ml, and after an interval of 2 weeks, a different point is injected with the same injection in the above mentioned parts, which can also produce highly effective immune serum. Common Problems I. Identification of results Determine the antibody potency of the obtained immune serum by biphasic agar diffusion test and identify the quality of the antibody in the immune serum by agar finger immunoelectrophoresis, which should produce a single precipitation arc. The specific steps of the identification method are described in the relevant sections. For more product details, please visit Aladdin Scientific website.
Sterilized saline IgG Alcohol Iodine Lanolin Paraffin Oil BCG FIA FCA-IgG
Scissors Tweezers Syringes Needles Weighing flasks Measuring cylinders Animal immobilizers Triangular flasks Surgical instruments Blood vessel clips Black wire Blood vessel release
It varies according to the nature of the antigen. The following is an example of the preparation of rabbit anti-human IgG immune serum for specific instructions.
1. Use scissors to cut off part of the rabbit's fur on both hind paws, and sterilize the skin with alcohol and iodine.
2. First immunization
Use a 2 ml syringe to aspirate 1 ml of Fuchs' complete adjuvant (FCA) emulsified antigen (human IgG), hereinafter referred to as FCA IgG liquid, and inject 0.5 ml into the subcutaneous of each paw on each side.
3. Second immunization
After an interval of 10-14 days, FCA-IgG was injected into the enlarged lymph nodes in both fossa and groin, 0.1 ml in each lymph node, and the rest was injected subcutaneously in the vicinity of the lymph nodes to make a total of 1 ml. If the lymph nodes were not enlarged or the enlargement was not obvious, it was injected directly into the subcutaneous part of both fossa and groin.
4. After an interval of 7-10 days, 0.5-1.0 ml of blood should be collected from the ear vein, the serum should be separated, and the antibody potency of the immune serum should be determined by biphasic agar diffusion test (i.e., blood test). The antibody potency should be at least 1:16 before the blood is released.
5. If the potency does not reach the required level, immunization can be performed by intra-auricular injection of antigenic liquid (human IgG) without adjuvant. That is to say, inject 3 times in 1 week, respectively 0.1, 0.3, 0.5 ml, and test the blood again at an interval of 1 week. If the potency reaches the requirement, the blood should be bled immediately. Alternatively, after the second immunization, the antigen (human IgG) emulsified with Fuchs' incomplete adjuvant (FIA) (FIA-IgG for short) can be immunized 1-2 times. The injection site, dosage and interval are the same as the second time, and then test the blood to measure the antibody potency, if the potency reaches the requirement, bleed immediately.
B. Bloodletting
1. Bloodletting method for heart
(1) The rabbit should be placed on its back, with its limbs tied to the animal fixation frame (or the limbs should be fixed by the assistant).
(2) Clip the fur from the left chest and sterilize the skin.
(3) Touch the left thumb to the sternal raphe, and place the index and middle fingers on the right chest to gently push the heart to the left and fix it on the left side of the chest. Then, touch the strongest part of the heart beat with the left thumb.
(4) Using a 50 ml syringe (with a 16-gauge needle attached), tilt the needle at a 45° angle and stab the heart at the site of the strongest heartbeat to draw blood.
(5) The extracted blood was immediately injected into a sterile triangular flask and the serum was separated after coagulation.
2. Carotid artery bloodletting
(1) The rabbit is fixed in supine position as above. The head is slightly lowered to expose the neck. Shave and sterilize the skin.
(2) The skin was incised along the midline of the neck for about 10 cm, and the subcutaneous tissue was separated until the sternocleidomastoid muscles on both sides of the trachea were exposed.
(3) Separate the loose tissue of the cervical triangle between the sternocleidomastoid muscle and the trachea, and free the common carotid artery after exposing it.
(4) Two black silk threads were inserted under the artery, placed distally and proximally. The wire at the distal end was ligated. The proximal end of the artery was clamped with a vascular clip.
(5) Using small pointed scissors, make a small cut in the arterial wall between the two wires and insert a plastic blood vessel. The proximal wire is then ligated and secured to the blood vessel to prevent the blood vessel from slipping out of place.
(6) Loosen the blood vessel clamp and let the blood flow into the sterilized triangular flask. Generally, a rabbit can be bled 80-100 ml.
Separation of serum
Place the blood in the triangular flask in a 37 ℃ incubator for 1 hour, and then place it in a 4 ℃ refrigerator for 3 to 4 hours. After the blood coagulates and the clot shrinks, use a capillary buret to suck up the serum. Centrifuge the serum at 3000 rpm for 15 minutes, take the supernatant and add preservative (0.01% thimerosal or 0.02% sodium azide, final concentration), and then store it in the refrigerator at 4 ℃.