Antiserum usually refers to polyclonal antibodies produced after artificial passive immunization, which is the main content of immunohistochemistry. Anti serum also becomes immune serum, mainly including preparation of immunogens, animal immunization, bloodletting, isolation of anti serum, analysis and identification of anti serum.
1、 Preparation of immunogens
Immunogen is a substance artificially made from antigens that can stimulate the body to induce humoral or cellular immunity. Immunogens are made from high-quality antigens and adjuvants. Commonly used adjuvants include Freund's incomplete adjuvant and Freund's complete adjuvant.
The specific preparation method of complete adjuvant: taking lanolin: paraffin oil1:3Proportional mixing high-pressure sterilization, add per milliliter1.5mg Made from BCG vaccine and used with the same antigen1:1Mix with equal volume and emulsify into a water in oil state.
Incomplete adjuvant is made by removing BCG vaccine from complete adjuvant.
2、 Animal immunity
Animals commonly used for animal immunization include rabbits (New Zealand big ear white, Japanese big ear white, etc.), sheep (Boer goats), guinea pigs, etc. Among them, rabbits have the advantages of good immune reactivity to antigens and suitable individual size, and are most commonly used. The IGG of rabbits is not divided into subtypes and is easy to manage in experiments. The animals used for immunization shall be age appropriate, healthy and free from infectious diseases, and the male is the best.
Immune sites: It is commonly used in the soles of the feet, inguinal lymph nodes, thigh muscles, subcutaneous tissue, etc. The combination of multiple sites has a good immune effect.
The dosage of immunization varies greatly depending on the size of the immunized animal, antigen molecule, and immunization regimen. According to the amount of antigen, it can be divided into trace, constant, and mass immunization methods. Trace immune antigens are at the microgram level; The amount of constant immune antigen is around 1-10mg; The dosage of a large amount of immune antigen is between 50-100mg; for mice it is in the microgram level, and for rabbits it is between 1mg and a few milligrams.
3、 Micro blood collection and potency testing
It is best to stimulate animals with 5-10mg of BCG vaccine first to stimulate the immune system, which is usually called basal immunity and can take 7-10 days to take effect. Next, inject the first dose to produce primary antibodies, with a higher concentration of IgM antibodies; Inject the second dose 2-4 weeks, and after 3-5 immunizations, collect a small amount of blood to test the potency. Collect the small amount of blood again 7-10 days after immunization. Rabbits use ear vein or artery for blood collection, while sheep can use ear vein or jugular vein for blood collection.
Rabbit blood collection: In winter, blood flow is slow. Rub the rabbit's ear with both hands until the arterial blood vessels in the middle of the ear are bulging. Use a needle to gently puncture the artery (be careful not to wipe alcohol or other liquids). At this point, the blood flows out in droplets and can be caught with an EP tube. In summer, due to active blood flow, venous blood collection can be used.
Sheep: Use scissors or an electric pusher to shave off the hair on the ears, rub the ears, wait for the blood vessels to swell, and use a syringe to extract.
The methods for detecting potency include simple ELISA, immunodiffusion, and one-way diffusion. Alternatively, other detection and analysis methods can be selected based on the purpose of preparing antibodies.
4、 Collection of immune serum
After achieving the expected goal of micro blood collection testing, the patient was euthanized by one-time bloodletting.
Rabbits and guinea pigs were euthanized by cardiac bleeding, while sheep were euthanized by venous bleeding.
Guinea pig blood collection 5-10ml; Rabbit, about 100ml, sheep, about 2500ml. Mice are bled by removing their eyeballs.
When collecting blood, pay attention to aseptic operation. After collection, place it in a large sterile container, let it solidify at room temperature for 30 minutes, then put it in a 37 degree freezer for solidification, and finally put it in a 4 degree freezer for full solidification. After complete coagulation, centrifuge at 3000-5000rpm for 20 minutes to obtain transparent serum.
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