Injecting the antigen into the animal body can stimulate the animal's B lymphocytes to transform into plasma cells and produce specific antibodies. When a large number of antibodies are accumulated in the animal's serum, its blood will be collected and the serum will be separated and analyzed, which is the antibody-containing immune serum, or antiserum. Preparation of specificity, high potency immune serum for the identification of microorganisms, diagnosis and treatment of infectious diseases, antigen analysis, identification of immunoglobulins and other protein identification and research have great use. Source: Laboratory Microbiology (Third Edition).
Operation method
basic program
Principle
The amount of antibody produced by an animal may vary depending on the species, age, nutritional status, and sensibility of the stimulated area of the animal, on the one hand, and on the other hand, it is also related to the type of antigen, the amount of injection, the route of injection, the number of injections, and the time between injections. When the amount of antigen is low to a certain limit, antibodies cannot be produced or cannot be measured by existing methods, but exceeding the maximum limit has an inhibitory effect on the production of antibodies. Above the minimum limit, antibodies increase according to the increase in the amount of antigen, and cease to increase when the maximum limit is reached. The amount of each type of antigen used is based on experience. The antigen in this experiment is bacterial cells, and it is appropriate to use 900 million bacteria per milliliter. After the initial injection of the antigen, antibodies can be found in the serum after a period of induction, and then gradually rise, usually in low amounts, and then gradually fall. However, when injected again, the amount of antibody rises rapidly to the highest level and is maintained for a long period of time. Therefore, the preparation of antibodies generally requires multiple injections of the antigen in order to obtain a highly effective immune serum. The most common route of injection is intravenous.
Materials and Instruments
E. coli slants cultured for 24 h Healthy male rabbits or non-pregnant healthy female rabbits around 2 kg Move 1. Antigen preparation 1.1 Aspirate 5 ml of sterilized 0.5% carbonate saline into an E. coli slant culture and wash down the moss. 1.2 Aspirate the washed off bacterial solution with a sterile capillary burette and inject into a small sterile test tube. 1.3 Place the test tube containing the bacterial solution in a water bath at 60°C for 1 h with occasional shaking. 1.4 Take a small test tube with the same mass as the turbidimeter, add 1 ml of bacterial solution, then add 4 ml of saline carbonate (or more, depending on the concentration of the original bacterial solution), mix well and then turbidimetry with the turbidimeter. Assuming that the turbidity is equal to that of the 3rd tube, the number of bacteria per milliliter of this bacterial solution is: 5 x 900 000 000 = 4 500 000 000 (4.5 billion) McFarland's Turbidimeter Preparation Method Use 10 test tubes of the same mass and size to add the drug according to the table below. 1.5 Dilution Dilute the solution with 0.5% carbonate saline to 900 million bacteria per ml. 1.6 Asepsis test Inoculate the diluted bacterial suspension with a small amount of broth medium, incubate for 24-48 hours, observe whether there is any bacterial growth, if there is no bacterial growth, put it in the refrigerator. 2. Preparation of immune serum 2.1 Injecting animals 2.1.1 Use a sterilized syringe and a No. 7 needle to extract the above prepared E. coli antigen (shake well before extraction), and inject into the ear vein of rabbits according to the dosage and schedule listed in the table below. 2.1.2 On the 14th day, collect 1 ml of blood from the ear vein, separate the serum, measure its agglutination potency, and collect a large amount of blood if it passes. 2.1.3 Collect blood on the 16th day. 2.2 Blood collection from the heart of rabbits (see "Animal blood collection test"). 2.3 Serum preparation 2.3.1 Transfer the collected blood into a large sterilized test tube (or flat dish), put it into the largest slant as possible, and then put it into a refrigerator at 4-6℃ after solidification, so as to let it precipitate out blood淸 naturally. 2.3.2 Separate the serum with a sterilized capillary burette. If there are red blood cells in the serum, centrifuge the serum to remove the red blood cells. The serum is then placed in sterilized vials and the potency of the antiserum is determined. 2.3.3 Preservatives are added so that the serum contains 0.01% thimerosal. 2.3.4 Seal the vials with wax or adhesive tape, label with the name of the antiserum, the agglutination potency, and the date, and place in the refrigerator. For more product details, please visit Aladdin Scientific website.
Thimerosal 0.5% carbonic acid saline
McFarland's turbidimeter Ethanol cotton Iodine cotton Sterilized dry cotton Sterilized pipettes Capillary burette Small test tubes Large test tubes Centrifuge tubes 2 ml and 20 ml syringes Old numbered 18-gauge and 24-gauge needles Sterilized vials Sterilized spouted bottle Centrifuge
