First, the purpose:
1. Understand the basic process of antiserum preparation:
2. Master the collection and preservation of antisera.
Second, the principle:
Antiserum is a serum containing a certain type of antibody molecules with specific immune function, and is generally an animal serum prepared after an animal is artificially injected with a certain type of antigen. Highly potent antiserum is used for research work and diagnosis and treatment of diseases.
Whether an antigen can cause antibody production depends on the presence or absence of an antigenic determinant on the surface of the antigen molecule, and on the other hand, the immune status of the body. When the above two conditions are met, antibody production will follow the antibody The general law of formation-the first reaction and the second reaction.
There are many types of antigens, including natural protein antigens, cellular antigens, synthetic antigens, and genetically engineered antigens. Animals immunized with different antigens have different specificities. Generally, complete antigen immunized animals require adjuvants, especially when using soluble antigens, in order to obtain highly effective antiserum; synthetic antigens and genetically engineered antigens and other hapten substances must be connected to protein carriers by artificial methods before being connected to The adjuvant can be used to immunize animals to obtain the desired immune effect. The use of adjuvants can increase the immunogenicity of the antigen and prolong the time the antigen remains in the body, thereby changing the original immunogenicity of the antigen.
3. Materials:
1. Metal number plate (marking the animal) or dye;
2. 1 ml, 5 ml, 20 ml syringes;
3. Blood collection needles (9 #, 12 #), card price needles (41/2
# — 51/2
#);
4. Sterilization plate, vaccine bottle, penicillin bottle, cork and straw, absorbent cotton;
5. BCG vaccine (75mg / ml);
6. Antigen (mouse serum);
7. Penicillin solution, streptomycin solution and 5% glucose solution;
8. Liquid paraffin (medical), wool wax (for adjuvant preparation);
9. Sterilized saline, alcohol for disinfection, 2.5% iodine;
10. 1.5% agar, glass plate, lunch box (moisturizing, moisturizing effect);
11. Experimental animals (rabbits).
4. Experimental steps
1. The choice of animals
Sheep, horses, chickens, monkeys, guinea pigs, and rabbits are commonly used immunized animals. In the experiment, when choosing animals, the relationship between antigen and animal species, the nature of antigen and animal type, the amount of immune serum required, Factors such as requirements and individual animals. Animals for immunization should be of suitable age and strong. It is best to be male. The most commonly used laboratory animal is the rabbit. Generally, choose the sex that is older than 6 months and breeds, weighs 2 to 3 kg, three healthy rabbits, fix the ears with metal number plates before immunization, or paint them with dye The foam is on the back of the animal, making a clear mark.
2. Antigen preparation
1) Antigen dilution: dilute the antigen to 2 mg / ml with sterile saline, add penicillin solution one day before immunization, so that each ml of antigen solution contains penicillin 1000 UI, streptomycin 1000 UI, and put at 4 ℃ overnight (usually not added Penicillin and streptomycin) were taken out the next day and mixed with an adjuvant to make an emulsion for animal immunization.
The injection dose of immunogen should consider its antigenic strength, molecular weight, animal's individual status and immunization time. For the immunization dose of pure soluble antigen, usually the first antigen dose is 50-100 μg / time for mice, 100-200 μg / time for rats, 100 μg-1 mg / time for rabbits, and 2 mg (approximately for haptens) 20 ~ 200μg), generally need to be mixed with equal amount of Freund's complete adjuvant. The booster dose is 1/2 of the first dose, usually with incomplete Freund's adjuvant or without adjuvant. If you need to prepare highly specific antiserum, you can use low-dose antigen short-range immunization method; on the contrary, if you want to obtain high-efficiency antiserum, it is appropriate to use large-dose antigen long-range immunization method.
2) Preparation of adjuvant and antigen emulsion
1 The preparation of adjuvant: Freund's complete adjuvant (Freund's complete adjuvant), the formula of each laboratory is different, the formula adopted in our laboratory is as follows:
Medical liquid paraffin
20ml
Lanolin 12g
Dissolve and mix in a water bath, divide it into penicillin vials, and tighten the stopper with gauze and thread. Sterilize at 8 pounds for 20 minutes and store at 4 ° C until use. When preparing the antigen emulsion preparation before immunization, live BCG vaccine (sometimes also used dead BCG vaccine) was used to replace dead tuberculosis bacteria, and 10 mg of live card was added to each milliliter of reagent, which was the complete Freund's adjuvant.
2 Preparation of antigen emulsion: There are generally two methods. First, the same amount of complete adjuvant (note that the adjuvant must be heated and melted in advance, but not more than 50 ℃) and the antigen solution are drawn into two 5ml syringes, in Between the 12 # needles of the two syringes, put a medical non-toxic plastic tube with a length of about 8 to 12 cm, and connect the two syringes together (the plastic tube must be immersed in alcohol for disinfection, take it out and use sterile saline After rinsing, connect with the needle of the syringe, the diameter of the plastic tube and the needle must be appropriate, not loose, it is better to tighten it), the needle is inserted into the plastic tube about 1 ~ 2cm, then the two people sit opposite and slowly push the needle core , So that the solution in the tube enters the plastic pipe to the opposite syringe, every time the needle core is pushed, the content of the tube must be pushed out, and the same operation is performed on the other side, so that the liquid in the tube is mixed back and forth until a water-in-oil emulsion is formed (Water-in -oil enulsion).
The second is that the same amount of adjuvant and antigen solution is poured into the bowl. After repeated grinding, the water-in-oil emulsion can also be formed. The main advantages of this method are fast and reliable. The deficiency is mainly due to excessive adhesion. Wasted adjuvant and antigen.
Whether the prepared emulsion forms a water-in-oil emulsion directly affects the immune effect, therefore, a quality check must be performed. The method of inspection is to drop the prepared emulsion on the surface of cold water (tap water), and the qualified emulsion is dropped on the water surface to keep the beads It is complete and not scattered. The unqualified person diffuses immediately after entering the water surface, and the oil on the water surface gradually expands. This must continue to operate until the quality is qualified.
But sometimes, due to the quality of the adjuvant, it is difficult to emulsify to pass. In this case, it is necessary to reconstitute the adjuvant.
3. Immunization route, dosage and immunization cycle
The route of antigen injection can be based on different antigens and test requirements. Intradermal, subcutaneous, intramuscular, intravenous or lymph nodes can be used to inject antigen for immunization. Generally, multiple intradermal or subcutaneous injections on the back, feet, around the lymph nodes, and behind the ears are often used. The interval between the first immunization and the second immunization is usually 2 to 4 weeks. The routine immunization program is antigen plus CFA subcutaneous multi-point injection for basic immunization; then the immunogen plus IFA is used for boosting immunization 2 to 5 times, with an interval of 2 to 3 weeks each time, subcutaneous or intraperitoneal injection to boost immunization.
1) Routine (multi-point intradermal injection) immunization:
1 The first immunization: the dose is 1.0 ml of antigen emulsion (containing 1 to 2 mg of antigen) per rabbit. The injection site is intradermal injection of 0.2 ml each of the two hind feet, and the remaining 0.6 ml is injected into the two sides of the spine, the neck, the groin, and the axilla in the vicinity of the lymph nodes, and each point can be injected with 0.05ml, divided into 12 points injection.
2 The second immunization: about 20 days after the initial immunization, the dose is 1.0ml / only (1-2mg / ml), plus the incomplete adjuvant of Fu's. The injection site was injected at multiple points under the skin of the abdomen, with 0.1 ml at each point.
3 The third immunization: within two weeks after the second immunization, the injection dose and location are the same as the second immunization, a little venous blood is drawn 7 to 14 days after the immunization, the serum is separated, and the blood test is used to determine the potency.
2) Lymph node immunity: The main advantage is that it can reduce the amount of antigen. The process is as follows:
1 Cardinal vaccine sensitization: Inject live live cardinal vaccine (75 mg / ml) 0.3 ml subcutaneously on each side of the forefoot. Observe the lymph nodes on both sides of the popliteal fossa for about 10 days, and they can generally swell as large as the size of broad beans. At this time, intralymphatic immunization injection can be performed.
2 The first immunization: after specifying the lymph nodes by hand, inject 0.25ml of antigen emulsion into the popliteal lymph nodes on both sides, 0.5ml in total for each rabbit; Add adjuvant antigen solution 1mg / 1ml.
3 After two weeks, you can inject again as the dose and route of the first immunization. Generally, you can titrate the titer by blood test before the third immunization injection. If the titer has reached the requirements, you do not need to perform the third immunization.
When conditions permit, animal feed management should be strengthened after immunization, such as feeding cooked soybeans.
3) Intraperitoneal immunity: generally without adjuvant, it is suitable for cellular or granular antigens, and those with strong antigenicity, such as rabbits or mice immunized with sheep red blood cells can use this method.
4. Blood test to determine antibody titer
Generally, a small amount of blood can be taken from the rabbit ear vein or the tail tip of the mouse 7 to 10 days after the third immunization, and the blood can be tested after separating the serum.
①The ring test, a more classic method, is seldom used at present. For example, if an antigen below 20 μg / ml can appear a “puppet†precipitation ring within 30 min, that is, if the antibody titer is above 1: 5000, it is qualified.
② Immunodouble-diffusion method, which is the most commonly used blood test method. The specific method is to add 10μl of diluted antigen (1mg / ml) in the middle hole, and the antiserum added in the clockwise direction around the holes is 1: 2, 1 : 4, 1: 8, 1:16, 1:32 and 1:64, (diluted with saline or PBS). Observe the result after incubating at 37 ℃ for 24h, and the blood value can be determined when the titer is above 1:16.
5. Bloodletting
The blood can be bled after passing the potency check. Animals should stop eating for 12 h before bleeding to reduce the fat content in the serum. If the immunized animal is to be kept, blood can be taken directly from the heart, or blood can be dripped from the vein of the ear or cut by heart puncture. After the blood is taken, an equal amount of 5% grape solution is slowly injected into the vein to make up for the blood loss. After the blood-sucked animals rest for 2 to 3 months, they can take blood after boosting again. If a bloodletting is planned to be fatal, the method of carotid bloodletting can be used. Various blood collection methods are as follows.
1) Blood collection from the heart: fix the animal in the supine position, use the index finger to detect the high cardiac momentum (on the left side of the sternum, between the third and fourth bones from the bottom, refer to the rabbit), cut a little hair, After sterilized with 2.5% iodine and 75% alcohol, it is 45 with the 9 # needle at the predetermined position with the chest. The horn penetrates the heart and moves the needle slightly up and down. After seeing the blood entering the syringe, the position of the syringe is fixed to take blood. A rabbit with a body weight of 2.5 kg can take 20 to 30 ml of blood at a time.
2) Blood dripping from ear veins: first cut the hair near the ear veins and wipe the skin with a sterile cotton ball (no alcohol disinfection to avoid hemolysis). Irradiate the ear with a desk lamp to expand the blood vessel due to the increase in temperature, and then use a sterile blade to cut a longitudinal incision of the ear vein around 0.5 cm. . Collect the outflow of blood with a sterile test tube or dish. If the incision clots and the blood flow is not smooth, you can use a sterile cotton swab to gently wipe out the blood clot outside the incision (be careful not to cause too much damage to the incision), the blood flow can continue to flow out until the required blood volume . After taking blood, use a sterile cotton ball to stop the bleeding. Blood from the ear vein can be taken for about 50ml at a time without the animal dying.
3) Carotid artery bleeding: The animal is fixed on the four limbs on its back, the neck is trimmed, and the skin of the anterior neck is cut longitudinally after sterilization. The length of the skin is about 10cm. Separation, that is, seeing the pulsating carotid artery. Carefully peel off the carotid artery and vagus nerve about 5 cm long, select the middle part of the blood vessel, and clamp the fascia around the blood vessel wall with hemostatic forceps. The distal end was ligated with silk thread, the proximal end was clamped with arterial forceps, and the cotton cotton ball was used to sterilize the blood vessel, and a V-shaped notch was cut with sterile scissors. Take a 2.5cm long and 1.6mm diameter plastic tube, cut one end into a needle-like bevel, insert this end into the carotid artery, put the other end into a 200ml sterile triangular bottle, then release the hemostatic forceps, and the blood flows in In the triangular flask, the animal bleeds to death, and the 2.5 kg rabbit can bleed 80 ～ 100ml.
6. Isolate serum
It must be performed under sterile conditions. After the blood collected in the dish or flask is coagulated, use a sterile dropper to remove the blood clot from the wall of the bottle in a sterile environment (such as an ultra-clean workbench) and place it at 37 ° C. After 1 ~ 2h, take it out and put it into 4 ℃ overnight, so that the serum can be fully analyzed (can not be frozen, otherwise hemolysis will occur), and the serum will be separated by centrifugal precipitation and put into a low temperature refrigerator for storage. It must be signed and qualified before use, and then packed and saved for future use.
7. Preservation of antiserum
1) After signing the qualified antiserum, add 1% thimerosal or 5% sodium azide at a ratio of 1: 100 to make the final concentration 0.01% or 0.05%, respectively.
2) Dispense antiserum into small tubes with a sterile dropper, each tube containing about 1ml.
3) Store below -20 ℃, can be stored for more than 2 years.
4) If conditions permit, serum can be freeze-dried and stored below 4 ° C for a longer period of time
Five, matters needing attention
1. There are individual differences in the immune response of animals. Some people even reported that the difference in antibodies produced by animals of different breeds can be as high as 500 times. Because some animals may produce antibodies with low titers or no antibodies, animals with good immune response can often provide 3-4 times higher antibodies than normal animals.
2. In order to prepare antiserum with high monovalent specificity, the higher the purity of the antigen used, the better. Therefore, in the process of purifying the antigen, try to remove possible miscellaneous proteins, so that it can save a lot of time to absorb the non-specificity in the antiserum Antibody.
3. To prepare highly effective antibodies, an adjuvant must be added (generally complete Freund's adjuvant). After adding the adjuvant to immunize the animal, the antibody titer can be increased by at least 5 times. However, it should also be considered that if trace amounts of contaminants are present in the antigen, even 0.005 mg, non-specific antibodies can be produced due to the adjuvant, so the adjuvant should be appropriately used according to the experimental needs and the purity of the antigen.
4. Whether the prepared antigen emulsion is water-in-oil emulsion should be checked, otherwise it will affect the immune effect, if it is not water-in-oil emulsion, it should be prepared again.
5. When the amount of antigen is too small and it is not easy to purify, the sediment line formed by the antigen antibody in the agar diffusion can be used to directly immunize the animal to prepare the antibody.
6. The titer of antiserum is generally expressed as the dilution of antiserum. There are many detection methods for serum titers and different sensitivities, so when expressing anti-serum titers, the detection method should be indicated. In addition, in the precipitation reaction, there is a large range of antigen and antibody ratios at the time of precipitation. For example, when using different concentrations of antigen to determine the titer, the results will be different. concentration.
7. The antiserum stored in separate packages should indicate the name, potency, date of preparation and packaging amount of the antiserum.
We are the supplier of nails including flat head nails, twist nails, bullet head nails, duplex nails, roofing nails, common nails, hardened grooved nails, concrete nails, pallet nails, spikes deck nails, staple nails, fence staple, plastic cap nails, coil nails, washer nails, made of carbon steel, Q195, Q235, stainless steel 304, stainless steel 316, etc. The nails is widely used in construction, furniture, Industry, agriculture.
Shank of nails:
Smooth Shank Nails: There are no deformations on the shank,making nails with a smooth shank the easiest to drive.Smooth shank nails offer the least pull-out resistance whencompared with spiral and ring shanks.
Concrete Nails: It is manufactured from ductile, high strength carbon steel and designed for hand hammer driving into medium strength concrete and solid block.
Roofing Nails: A nail used for attaching paper or shingles to roof battens or sheathing; usually with a large head.
Annular Ring Shank: Annular threads or ''rings" areformed on the shank to increase withdrawal capacity. The[rings" create an interlock between the shank of the nailand the wood, providing superior holding power.
Spiral Shank: A spiral ''thread" on the shank causes thenail to spin during installation, creating a thread-like interlockwith the wood, which increases withdrawal capacity.Spiral-shank nails are designed to drive easier into harderwoods and dense materials while still providing increasedwithdrawal resistance.
Packing:
1kg/box, 5kg/Box,20kg/Carton, 30kg/Carton; or as your requirements.
Iron Steel Common Nail, Concrete Nail for construction, Galvanized Roofing Nail
DINGZHOU TIAN YILONG METAL PRODUCTS CO., LTD. , https://www.wiremeshsolution.com