Antibody preparation method and principle: preparation of antiserum

1. Animals used for immunization

Animals used for immunization include mammals and poultry, mainly sheep, horses, rabbits, monkeys, pigs, guinea pigs, chickens, etc. The laboratory commonly used are rabbits, goats and guinea pigs. The choice of animal type is mainly based on the biological characteristics of the antigen and the amount of antiserum to be obtained. For example, anti-r-immunoglobulin antiserum is generally prepared. Multi-purpose rabbits and goats are used because the animals respond well and can provide a sufficient amount of serum. The immunized animals should be age-appropriate, strong, and free from infectious diseases, preferably // males. In addition, great care should be taken in animal feeding to eliminate individual differences in animals and the effects of death during the immunization process. If rabbits are used, it is best to use pure-bred New Zealand rabbits, a group of three, the weight of rabbits should be 2 ~ 3kg.

2. Immunization

There are a variety of immunization routes, such as intravenous, intraperitoneal, intramuscular, intradermal, subcutaneous, and intra-lymph node injections. Generally, subcutaneous or back multi-point intradermal injections are usually used, and each point is injected with about 0.1ml. The choice of route depends on the biological and physical and chemical characteristics of the antigen, such as hormones, enzymes, toxins and other biologically active antigens, intravenous injection is generally not suitable.

3. Adjuvant

Because different individuals have different reactivity to the same antigen, and the ability of different antigens to generate immune responses is also strong or weak, so often when the antigen is injected, an antigenic substance that can enhance the antigen is added to stimulate the body to produce a stronger immune response , This substance is called immune adjuvant.

In addition to prolonging the residence time of the antigen in the body and increasing the stimulation effect of the antigen, the adjuvant is more important, it can stimulate the reticuloendothelial system, increase the number of immune active cells involved in the immune response, and promote the interaction between T cells and B cells. Thereby enhancing the body's cellular immunity to antigens and the production of antibodies.

The commonly used adjuvant is Freund adjuvant. Its components are usually 1 part of lanolin and 5 parts of paraffin oil. The ratio of lanolin to paraffin oil can be adjusted to 1: 2-9 (V / V), this is incomplete Freund's adjuvant, and adding 1 to 20 mg of BCG vaccine per ml of incomplete adjuvant becomes a complete adjuvant.

Preparation method: Place the lanolin and paraffin oil in the container according to the proportion, mix them with ultrasonic waves, autoclave, and store at 4 ℃ for later use. Before immunization, take equal volume of complete or incomplete adjuvant and immunogen solution, mix it with a shaker to make it milky. You can also take the required amount of adjuvant in the mortar before grinding, and evenly add it while grinding. Volume of antigen solution (including BCG vaccine 3 ~ 4mg / ml or not), after adding, continue to grind into an emulsion, drip on ice water within 5 ~ 10min until no diffusion. In order to avoid the loss of antigen, one syringe can be used to hold the antigen solution and the other can be used to connect the adjuvant. The two are connected by a polyethylene plastic tube, and then the two are repeatedly pumped back and forth, which can be completely emulsified after about tens of minutes. After passing the inspection, one of the syringes is used for injection.

4. Immunization method

Antigen dose, the first dose is 300 ~ 500μg, the booster dose is about 1/4 of the first dose. Boost immunity every 2 to 3 weeks. Incomplete adjuvant is used for booster immunization. Pertussis vaccine 0.5ml is injected subcutaneously during the first immunization. Pertussis vaccine is not necessary for booster immunization.

Two weeks after the second booster immunization, 2 to 3 ml of blood was taken from the marginal ear vein to prepare serum and detect antibody titer (see later). If the expected titer is not reached, booster immunization will be required until it is satisfactory (Figure 2-3). When the antibody titer reaches the expected level, anti-serum can be prepared by bleeding.

5. Collection and storage of antiserum

Rabbits are the most commonly used animals to produce antibodies, so here we mainly discuss the collection of rabbit blood. For larger animals such as sheep, blood is taken through the jugular vein and arteries. For small animals such as rats, blood can be obtained from the relevant information. There are two ways to draw rabbit blood, one is bleeding from the ear vein or ear artery, the other is blood from the carotid artery, and blood can also be collected from the heart. When taking blood from an artery or vein, put the rabbit in a specially made wooden box or cage, with the ear exposed outside the box (cage), or another person can catch the rabbit. Cut the hair on the edge of the ear and apply a little xylene to the auricle. After 30 seconds, the ear blood vessels dilate and congest. Pull the tip of the ear gently with your hand, and use a single-sided razor or sharp surgical blade to quickly cut the artery or vein, and the blood will flow out, and 30 to 40 ml can be collected each time. Then use cotton balls to compress the hemostasis, wash the xylene after clotting. After two weeks, bleeding can be done on the other ear. This method can repeatedly bleed many times. When bleeding blood from the carotid artery, lie on the back of the rabbit, fix it to the rabbit table, cut the hair of the neck, cut the skin, expose the carotid artery, intubate, and bleed. Strictly follow the sterility requirements during the bloodletting process.

The collected blood is placed at room temperature for about 1h. After coagulation, the serum is precipitated at 4 ° C overnight (do not freeze) and centrifuged at 4000rpm for 10min. Under aseptic conditions, aspirate the serum and divide it into aliquots (0.05-0.2ml), store it in the refrigerator below -40 ℃, or store it in 4 after lyophilization. C refrigerator to save.

6. Evaluation of antiserum quality

During immunization, not only different animals, but also the same animal's antiserum titer, specificity, affinity, etc. may change at different times, so blood tests must be performed frequently. The antiserum obtained can only be used after a thorough evaluation of the antiserum's potency, specificity, affinity, etc.

1. Potency

The titer of antiserum refers to the concentration or content of antibodies contained in the serum. The method of potency determination is usually radioimmunoassay, which is applicable to all antibodies. Certain antibodies produced by large molecule (eg protein) antigens can be measured by double diffusion and other methods. The titer determined by the former is extremely accurate. The latter is much rougher.

(1) Radioimmunoassay

Anti-serum of different dilutions was mixed with high-quality labeled antigen, and after 24 hours of incubation, the binding rate was determined. Serum dilution and binding rate of 50% are usually used as titers. If the dilution rate of a certain antiserum when the binding rate is 50% is 1: 15000, the titer of the serum is 1: 15000. The titer of antiserum is not only determined by the nature of the antiserum, but also affected by the quality of the labeled antigen, the incubation time, the composition of the diluent used and its pH and other factors, it must be noted in the work.

(2) Bidirectional diffusion method

Macromolecular antigens and antibodies are used to spread on agar plates, and the two produce precipitation lines at the intersection to observe and determine whether there are antibodies and their concentrations in the antiserum.

Preparation of agar plates: 100ml of phosphate buffer pH 7.1 was added to 15g of agar, heated in a water bath, stirred to completely dissolve the agar, filtered with gauze while hot, and when the solution was cooled to about 65 ℃ Sodium azide (NaN3), so that its concentration in the solution is 0.1%. Use a pipette to put the agar on a clean plate or glass slide, about 3mm thick. After it cools and completely solidifies, punch the hole with a puncher. Add appropriate amount of antigen (capacity is 50μl) into the central well, and add 50μl of 1: 2, 1: 4, 1: 8, 1:16, 1:32 and undiluted antiserum to each surrounding well, and incubate at 37 ℃ for 24h , Observe whether there is a precipitation line to determine the dilution of serum.

2. Specificity determination

The specificity or specificity of antiserum refers to the ability of antiserum to recognize corresponding antigens and similar antigenic substances. Good specificity is the ability of antiserum to recognize. Generally, specificity is expressed in terms of cross-reactivity. The low cross-reactivity rate indicates that the specificity of the antiserum is good, otherwise the specificity is poor. The cross-reaction rate is generally judged by the competition inhibition curve. Competitive inhibition curves were made with different concentrations of antigen and approximate antigen substances, and their respective binding rates (B / T or B / B0) were calculated, and their respective concentrations at IC50 were calculated, and the cross reaction rate was calculated according to the following formula.