First, the preparation of antiserum
With good quality antigens, you must also choose the appropriate immune pathway to produce good quality (high specificity and high titer) antibodies.
(1) Animals used for immunization
The animals used for immunization include mammals and poultry, mainly sheep, horses, rabbits, monkeys, pigs, guinea pigs, chickens, etc., and commonly used in the laboratory are rabbits, goats and guinea pigs. The choice of animal species 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, and rabbits and goats are often used. The animals respond well and can provide a sufficient amount of serum. Animals that are immunized should be of appropriate age, robust, and free of infectious diseases, preferably // males. In addition, animal feeding should be paid attention to in order to eliminate individual differences in animals and the effects of death during immunization. If rabbits are used, it is best to use purebred New Zealand rabbits, a group of three, and the rabbits should weigh 2 to 3 kg.
(two) immune pathway
There are various ways of immunization, such as intravenous, intraperitoneal, intramuscular, intradermal, subcutaneous, intralymphatic injection, etc., generally used subcutaneous injection of subcutaneous or back, about 0.1ml per point. The choice of pathway depends on the biological characteristics and physical and chemical properties of the antigen, such as biological active antigens such as hormones, enzymes, toxins, etc., and intravenous injection is generally not suitable.
(three) adjuvant
Because different individuals have different responsiveness to the same antigen, and different antigens have strong and weak immune responses, antigenic substances that enhance antigens are often added at the same time as the antigen is injected to stimulate the body to produce a strong immune response. This substance is called an immunological adjuvant.
In addition to prolonging the retention time of the antigen in the body and increasing the stimulation of the antigen, the adjuvant mainly stimulates the reticuloendothelial system, increases the number of immunoreactive cells involved in the immune response, and promotes the interaction between T cells and B cells. Thereby enhancing the cellular immunity and antibody production of the antigen.
The commonly used adjuvant is Freund adjuvant. The composition is usually 1 part of lanolin, 5 parts of paraffin oil, and the ratio of lanolin to paraffin oil can be adjusted to 1:2～9 (V). /V), this is an incomplete Freund's adjuvant, and 1 to 20 mg of BCG is added as a complete adjuvant in incomplete adjuvant per ml.
Preparation method: The lanolin and paraffin oil are placed in a container in proportion, mixed with ultrasonic waves, autoclaved, and stored at 4 ° C for use. Before immunization, take an equal or incomplete adjuvant and mix with the immunogen solution, mix it with a shaker to form a milky shape, or use the adjuvant to grind it in the mortar before immunization, evenly add it after grinding. The volume of the antigen solution (in which 3 to 4 mg/ml is added or not), after the addition, continue to grind into an emulsion, and drip on the ice water for 5 to 10 minutes without any diffusion. In order to avoid loss of antigen, a syringe can be used to mount the antigen solution, and the other syringe is filled with an adjuvant. The two are connected by a polyethylene plastic tube, and then the two are repeatedly sucked back and forth, and can be completely emulsified after about several minutes. After the test is passed, one of the syringes is used for injection.
(4) Immunization methods
The dose of the antigen is 300-500 μg for the first dose, and the dose for boosting is about 1/4 of the first dose. Intensive immunization every 2 to 3 weeks. Intensive immunization with incomplete adjuvant, subcutaneous injection of pertussis vaccine 0.5ml for the first immunization, no need to inject pertussis vaccine when boosting immunization.
Two weeks after the second booster immunization, 2 to 3 ml of blood was taken from the ear vein to prepare serum and the antibody titer was detected (see later). If the expected titer is not achieved, booster immunization is required until satisfactory (Figure 2-3). When the antibody titer reaches the desired level, the antiserum can be prepared by exsanguination.


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