Polyclonal Antibody Preparation Experiment
Polyclonal Antibody Preparation Experiment
Antigens are usually composed of multiple antigenic determinants, and the antibody produced by a B-lymphocyte receiving an antigen stimulated by one antigenic determinant is called a monoclonal antibody. When the organism is stimulated by a variety of antigenic determinants, a wide range of monoclonal antibodies are produced accordingly, and when these monoclonal antibodies are mixed together, they are polyclonal antibodies, and the antibodies produced in the organism are polyclonal antibodies; in addition to the diversity of antigenic determinants, the same type of antigenic determinants can stimulate the organism to produce five types of antibodies, such as IgG, IgM, IgA, IgE, and IgD.
Operation method
Fuchs' adjuvant immunization
Principle
When an antigen is injected into an experimental animal, a series of antibody-producing cells will bind to the antigen in varying degrees, and produce different types of antibodies in the blood after being stimulated by the antigen. This type of antibody produced by the stimulation of one type of antigen is called a polyclonal antibody. Different antibody molecules in polyclonal antibodies can bind to different parts of the surface of the antigen molecule - antigenic determinants - with different affinity. Introduction of the antigen into a sensitive animal stimulates the reticuloendothelial cell system, especially lymphocytes in the lymph nodes and spleen, to proliferate. As shown in the figure, there is a marked difference in the response of experimental animals to primary and secondary immunization. In general, the primary immune response tends to be weak, especially against readily metabolizable, soluble antigens. Antibodies are observed in the serum about 7 days after the first injection, but the concentration of antibodies remains at a low level, and the titer of antibodies reaches a maximum in about 10 days. However, the results of the secondary immune response to the same antigen are significantly different, as the rate of antibody synthesis is significantly higher and the retention time is longer than in the initial immune response. The kinetics of the immune response depends on the antigen and the type of animal immunized, but the relationship between the primary and secondary immune responses is an important feature of the immune response. Three or more injections of antigen produce a response similar to that of the secondary response: a marked increase in antibody titer and a change in the type and nature of the antibody in the serum, a change known as maturation of the immune response, which is of great practical importance. Antibodies with high affinity are usually produced 4-6 weeks after antigen injection.
Materials and Instruments
New Zealand rabbit Move I. Preparation of experimental materials Caveat Rabbits need to be kept immunized and bled for 9 weeks. For more product details, please visit Aladdin Scientific website.
Incomplete Freund's Adjuvant (IFA) Complete Freund's Adjuvant (CFA) Protein KLH-coupled peptide Physiological saline
Syringe
1. Immunized animals: two New Zealand rabbits.
2. Adjuvant: Complete Freund's adjuvant (CFA) first, followed by incomplete Freund's adjuvant (IFA).
3. Immunogen: protein or KLH-coupled peptide. Immunize with 100-200 μg of immunogen at a time.
Experimental procedure
1. Immunization
The immunogen was diluted with saline and then mixed 1:1 with the corresponding adjuvant. The antigen and adjuvant are thoroughly mixed to form a stable emulsion, which is injected subcutaneously under the skin around both shoulders and intramuscularly in the hind thighs of the rabbit. Approximately 1/4 of the immunogen is used in each area. This allows the immunogen to persist and thus increase the immune response.
2. Blood sampling
Blood was taken from the ear artery of the rabbit with a 19-gauge needle and the serum was analyzed overnight at room temperature.
3. Periodic immunization and bloodletting
(1) Week 0
Blood was collected in 2 ml (to obtain 0.5-1 ml of pre-immune serum), and rabbits were immunized with 200 μg of antigen mixed with complete Freund's adjuvant (CFA).(2) Week 2
Rabbits were immunized with 200 μg of antigen mixed with complete Freund's adjuvant (CFA).
(3) Week 4
Rabbits were immunized with 100 μg of antigen mixed with incomplete Freund's adjuvant (IFA).
(4) Week 5
Take 20-30 ml of test serum, appropriate cells or tissues for ELISA and WB, and immunize rabbits with 100 μg of antigen dissolved in saline.
(5) Week 6
If the test is positive the first time 20-30 ml of blood is collected and 100 μg of antigen dissolved in saline immunizes the rabbit; if the last test was negative, appropriate cells or tissues are taken for ELISA and WB.
(6) Week 7
A second blood collection of 20-30 ml of 100 μg of antigen dissolved in saline immunizes the rabbit; if the test remains negative, the continuation of this program needs to be discussed.
(7) Week 8
A third blood collection of 20-30 ml of 100 μg of antigen dissolved in saline immunized the rabbits.
(8) Week 9
The final blood release was 20-30 ml, and the mixed blood samples were purified, yielding an average of 100-150 mg of antibody per purification, and finally, quality tests such as ELISA and WB were performed.