Serological technique

Master the principles, methods and applications of classic in vitro antigen-antibody reactions.

Section 1 Preparation of Immune Serum

The preparation of immune serum is a commonly used immunological experimental technique. High-efficiency, high-specificity immune sera can be used as reagents for immunological diagnosis (such as for the preparation of immunolabeled antibodies, etc.), and can also be used for specific immunotherapy. The titer of immune serum depends on the immunoreactivity of laboratory animals and the immunogenicity of antigens. If a highly responsive organism is stimulated with an immunogenic antigen, a highly potent immune serum can often be obtained. When immunizing with a weak immunogenic antigen, an adjuvant must be added to enhance the immunogenicity of the antigen. The specificity of immune serum mainly depends on the purity of the antigen for immunization. Therefore, if you want to obtain a highly specific immune serum, you must first purify the antigen. In addition, the dose of antigen, the route of immunization and the time interval of antigen injection are also important factors that affect the titer of immune serum and should be paid attention to.

Basic principles Immunogenic antigens can stimulate the body's corresponding B cells to proliferate, differentiate into plasma cells and secrete specific antibodies. Since different determinants on the surface of an antigen molecule are recognized by B-cell clones with different specificities, the antibody produced by stimulating the body with an antigen is actually a mixture of antibodies against different determinants on the surface of the antigen molecule (ie polyclonal antibodies ).

In this experiment, the anti-human IgG immune serum was prepared by using the antigen (human IgG) emulsified by Freund's complete adjuvant (FCA). Students are required to initially learn the basic methods of preparation of immunogens and adjuvants and preparation of immune sera, understand their significance and application, and be familiar with the basic knowledge of animal experiments.

Materials 1. Animals: Healthy adult rabbits, male, weighing 2 to 3 kg.

2. Equipment: Scissors, tweezers, syringes (2ml, 50ml) with needles (No. 9 and No. 6), weighing bottles (10ml), measuring cylinders, animal holders, sterilized triangular flasks (200ml), a set of surgical instruments, Vascular clamps, black silk thread, plastic blood vessels, etc.

3. Reagents: sterilized physiological saline; purified human IgG (10mg / ml); sterilized alcohol and iodine; lanolin; paraffin oil; live cardine vaccine (BCG) (75mg / ml); Freund ’s incomplete adjuvant ( FIA) Preparation: Weigh 10 grams of lanolin, add 40 ml of high-quality paraffin oil drop by drop, grind while dropping, and distribute in vaccine bottles (10 ml per bottle), autoclave (8 pounds for 20 minutes) and save for later use. Preparation of Freund's complete adjuvant emulsified antigen (FCA-IgG): warm FIA (60 ° C for 30 minutes), pipette 3ml into a mortar, add 0.5ml of live BCG (75mg / ml) and purified human IgG2 dropwise. 5ml (2.4mg / ml) squeeze_ drop and grind until a homogeneous emulsion is formed. Take 1 drop on the cold water surface and do not spread it to pass.

method:

1. Immunization method: It varies according to the nature of the antigen. In the following, the preparation of rabbit anti-human IgG immune serum is taken as an example for specific explanation.

(1) Use scissors to cut off part of the rabbit hair on the hind paws of the rabbit, and disinfect the skin with alcohol and iodine;

(2) The first immunization: 1ml of Freon's complete adjuvant (FCA) emulsified antigen (human IgG) (hereinafter referred to as FCA-IgG) solution was sucked with a 2ml syringe, and 0.5ml was injected subcutaneously on each foot.

(3) Second immunization: After 10-14 days interval, FCA-IgG was injected into the enlarged lymph nodes on both sides of the fossa and groin, each lymph node was injected with 0.1ml, and the rest was injected into the subcutaneous lymph nodes for a total of 1ml. If the lymph nodes are not enlarged or the enlargement is not obvious, directly inject it under the skin on both sides of the nest and the groin.

(4) After an interval of 7-10 days, 0.5 to 1.0 ml of blood was collected from the ear vein, the serum was separated, and the antibody titer of the immune serum (ie, blood test) was measured by a two-phase agar diffusion test. Blood can only be bleed when the potency should be at least 1:16 or more.

(5) If the titer does not meet the requirements, the antigen solution (human IgG) without adjuvant can be injected into the ear vein for immunization. That is, three injections within a week, respectively, 0.1, 0.3, 0.5ml. Retest blood at 1 week intervals. If the potency meets the requirements, blood should be bleed immediately. In addition, after the second immunization, the antigen (human IgG) emulsified with Freund's Incomplete Adjuvant (FIA) (referred to as FIA-IgG) may be immunized for 1-2 times. The injection site, dose and interval are the same as the second time, and then test the blood to test the antibody titer, if the titer reaches the requirement, immediately bleed.

2. Bloodletting:

(1) Blood method for heart

(1) The rabbit is on his back, his limbs are bound to the animal's fixed frame (or his assistant can hold the limbs to fix it);

(2) Cut the rabbit hair of the left chest and disinfect the skin;

(3) Touch the xiphoid of the sternum with the left thumb, place the index finger and middle finger on the right chest, gently push the heart to the left, and fix the heart on the left chest. Then, touch the strongest part of the heart with your left thumb;

(4) Use a 50ml syringe (connected to the 16-gauge needle), tilt the needle at a 45 ° angle, puncture the heart with the strongest heartbeat and draw blood to kill;

(5) Immediately inject the drawn blood into a sterile Erlenmeyer flask and separate the serum after coagulation.

(2) Carotid bloodletting method

(1) The rabbit is supine as above. 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 and separate the subcutaneous tissue until the sternocleidomastoid muscles on both sides of the trachea are exposed;

(3) Isolate the loose tissue in the cervical triangle between the sternocleidomastoid muscle and the trachea, and free it after exposing the common carotid artery;

(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 proximal arteries are clamped with vascular clamps;

(5) Use pointed small scissors to make a small cut in the arterial wall between the two silk threads and insert the plastic tube. Then ligature and fix the proximal silk thread on the blood vessel to prevent the blood vessel from slipping;

(6) Loosen the blood vessel clamp and allow blood to flow into the sterilized Erlenmeyer flask. Generally a rabbit can bleed 80 ～ 100ml.

3. Separation of serum: Place the blood in the Erlenmeyer flask in a 37 ° C incubator for 1 hour, and then place it in a 4 ° C refrigerator for 3 to 4 hours. After the blood clot contracts, use a capillary pipette to draw the serum. Centrifuge at 3000 rpm for 15 minutes, take the supernatant and add preservatives (0.01% thimerosal or 0.02% sodium azide, final concentration), store it in a refrigerator at 4 ° C for later use.

Results identification:

The antibody titer of the obtained immune serum was determined by the two-phase agar diffusion test, and the quality of the antibody in the immune serum was identified by agar finger electrophoresis, and a single precipitation arc should be generated. See the relevant section for the specific steps of the identification method.

Precautions:

1. The antibody reactivity of experimental animals for immunization varies greatly, so at least two animals should be selected for immunization. In addition, pregnant animals cannot be used.

2. The antigen for immunization must be fully emulsified by FCA or FIA before injection, otherwise it will obviously affect the immune effect of the antigen. The above method for preparing emulsified antigen is time-consuming and laborious. It is prepared by H-81 micro-oscillating mixer method or three-chamber tube grinding method. The former is to mix the antigen solution and the adjuvant in proportion, and put it on the mixer to vigorously oscillate (frequency 2900 rpm, amplitude 6mm); the latter is to mix the adjuvant and antigen solution for immunization in proportion, Inhale into the syringe and then connect the syringe to the three-lumen tube to repeatedly suck and grind. These two methods can fully emulsify the antigen in about 1 hour.

3. On the one hand, adjuvant can improve the effect of specific immune response and obtain high-efficiency immune serum. However, if the antigen is not pure, it can cause extremely trace contaminants (0.005mgN) in the antigen to produce antibodies, which leads to the purity of immune serum affected. In addition, some experimental animal strains are allergic to BCG, especially guinea pigs followed by rabbits. When the complete adjuvant is injected again, it can cause allergies and lead to immune failure. For this reason, the second immunization injection should reduce the content of BCG in the adjuvant or switch to incomplete adjuvant to reduce and prevent the occurrence of allergy.