Antigens are usually composed of multiple antigenic determinants, and the antibody produced by a B lymphocyte stimulated by one antigenic determinant is called a monoclonal antibody. Multiple antigenic determinants stimulate the body, resulting in the production of various monoclonal antibodies. These monoclonal antibodies are mixed together to form polyclonal antibodies, and the antibodies produced within the body are called polyclonal antibodies; In addition to the diversity of antigenic determinants, the same antigenic determinant can also stimulate the body to produce five types of antibodies, including IgG, IgM, IgA, IgE, and IgD. Antigens stimulate the body, producing immunological reactions. A group of immunoglobulin that is synthesized and secreted by plasma cells of the body and has specific binding ability to antigens is called immunoglobulin. This immunoglobulin that has specific binding ability to antigens is called antibody.
一、organizational structure
The molecular structure on the antigen that can cause the body to produce antibodies is called the antigenic determinant cluster. There can be several different antigenic determinants on an antigen, which leads to the production of several different antibodies in the body, ultimately resulting in plasma cells producing antibodies. A plasma cell population that only acts on one antigenic determinant is a pure line, which is called Clone in English. The specific antibody produced by a clone is called a monoclonal antibody. Monoclonal antibodies can bind specifically to a single antigenic determinant, just like a missile hitting its target with precision. On the other hand, even for the same antigenic determinant, antibodies can be produced by several clones in the body, forming a mixture of several monoclonal antibodies, known as polyclonal antibodies.
二、Antibody classification
An antigen is usually composed of multiple antigenic determinants, and the antibody produced by a B lymphocyte receiving the antigen stimulated by one antigenic determinant is called a monoclonal antibody. Multiple antigenic determinants stimulate the body, resulting in the production of various monoclonal antibodies. These monoclonal antibodies are mixed together to form polyclonal antibodies, and the antibodies produced within the body are called polyclonal antibodies; In addition to the diversity of antigenic determinants, the same type of antigenic determinant can also stimulate the body to produce five types of antibodies, including IgG, IgM, IgA, IgE, and IgD. Polyclonal antibodies are a group of immunoglobulins secreted by plasma cells of an organism stimulated by heterologous antigens (macromolecular antigens, hapten conjugates) to produce an immune response. Polyclonal antibodies are widely used in research and diagnosis due to their ability to recognize multiple antigenic epitopes, induce precipitation reactions, short preparation time, and low cost.
三、Antigen preparation
There are many substances that can serve as antigens. In general scientific research experiments, antigens commonly used include conjugated peptides, conjugated small molecules or compounds, natural or recombinant proteins, and so on.
For soluble antigens, in order to enhance their immunogenicity, change the type of immune response, and save antigens, adjuvants are often used to stimulate the body to produce a strong immune response.
四、Adjuvant application
1．The types of adjuvants commonly used in practice include aluminum hydroxide gel, alum, Freund's adjuvant, liposomes, and paraffin oil. There are also methods such as using Bacillus subtilis to divide into Bacillus subtilis, Corynebacterium diphtheriae, and Corynebacterium parvum.
⑴ Incomplete Freund's adjuvant (IFA)
1 part lanolin, 5 parts paraffin oilMix and store after high-pressure sterilization. Heat and melt during use, cool to around 50 ℃, and emulsify with antigen.
⑵ complete Freund's adjuvant,CFA
Freund's incomplete adjuvant 10ml
BCG vaccine 10mg~200mg can be inactivated at 100 ℃ for 10 minutes.
It is best to use Freund's complete adjuvant during the initial immunization to stimulate the body to produce a strong immune response. When immunizing again, complete adjuvant is generally not used, but Freund's incomplete adjuvant is used. But when studying mycobacteria and related antigens, Freund's complete adjuvant is generally not used to avoid interference from BCG.
⑶ Liposomes：are artificially prepared lipid like small spheres composed of one or more lipid bilayers resembling cell membranes. This structure enables them to carry various hydrophilic, hydrophobic, and amphoteric substances, which are encapsulated in the aqueous phase inside the liposome, inserted into the lipid bilayer, or adsorbed and attached to the surface of the liposome, playing a significant role in immune enhancement.
⑷ Oil adjuvant ：Both vegetable oil and mineral oil can be used, including soybean oil, peanut oil, rapeseed oil, etc. The most widely used mineral oil. No.10 white oil (paraffin oil) 100ml
2g aluminum stearate
Siben 80 6ml
Mix and heat to melt, then pack separately. Emulsify according to the following formula when used.
3 parts of oil phase
1 part of aqueous phase (with 4% Tween-80 added)
First stir the oil phase, then slowly add the water phase for emulsification. Siben is an oil dispersant and Tween is a water dispersant, both of which are beneficial for emulsification.
5、Emulsification effect
The process of mixing antigens with adjuvants is called emulsification. There are many methods of emulsification, and mortar emulsification can be used; Can be directly emulsified on a vortex oscillator; Can be emulsified using a tissue grinder. When used in small quantities, especially when Freund's adjuvant is emulsified with antigens, a syringe is often used for emulsification. Two syringes are used, one for inhaling the antigen solution and the other for inhaling the adjuvant. The heads of the two syringes are connected by a hose, and it is important to tighten them tightly before suctioning back and forth. When emulsifying in large quantities, a colloid mill can be used.
The sign of good emulsification is that a drop of emulsifier is dropped into water and appears spherical without dispersing. If there is flat diffusion, it means that the emulsion is not well emulsified. Emulsified substances that have been stored for a period of time (within the shelf life) and show oil-water stratification are also signs of poor emulsification. The immune pathway, frequency, and interval time are determined based on the nature, immunogenicity, and animal immunoreactivity of the antigen. The immune pathways include subcutaneous injection, intradermal injection, intramuscular injection, intravenous injection, intraperitoneal injection, and lymph node injection. If the amount of antigen is small, adjuvants are generally used for multi-point injection within or around lymph nodes, or in the foot or palm, or intradermal or subcutaneous injection. If the amount of antigen is large, subcutaneous, intramuscular, or even intravenous injection can be used. Intradermal and subcutaneous injections with adjuvants are usually administered every 2-4 weeks. Subcutaneous or intramuscular injection without adjuvant, usually with an interval of 1-2 weeks; Muscular or venous immunity, with an interval of about 5 days. Various injection routes can be combined and applied to achieve the desired potency.
6、Immune animals
The animals used for immunization are mainly mammals and poultry, often including rabbits, sheep, goats, horses, mules, guinea pigs, and mice. The selection of animals is often determined by the use and amount of antibodies, as well as the nature of the antigen. To obtain a large amount of antibodies, large animals are often used; If antibodies directly labeled for diagnosis are obtained, use the animal directly; To obtain indirectly labeled diagnostic antibodies, heterologous animals must be used to prepare antibodies; If it is difficult to obtain antigens and the required amount of antibodies is small, pure strain mice can be used for preparation; The antibodies commonly used in laboratories are often prepared from rabbits and sheep.
It is best to choose healthy male animals of appropriate age for immunization. Female animals, especially pregnant animals, are not suitable for preparing immune antibodies, and sometimes do not even produce antibodies. Due to individual differences in immune response, multiple animals should be selected simultaneously for immunization. Antigens are diverse and vary greatly. In terms of its chemical composition, there are protein antigens, lipid antigens, polysaccharide antigens, and nucleic acid antigens. In terms of antigenicity, there are complete antigens and incomplete antigens. In order to obtain better anti serum, it is best to use protein antigens. If preparing anti serum for screening bacterial expression cDNA libraries or immunoblotting, it is best to use degradable protein antigens. The strength of immunogenicity of different antigens varies depending on their molecular weight, chemical active groups, three-dimensional structure, physical shape, and diffusion rate.
The immune dose of antigens varies depending on the type of animal administered, the immune cycle, and the required antibody characteristics. Low dose may not elicit sufficient immune stimulation, while excessive immune dose may lead to immune tolerance. Within a certain range, the potency of antibodies increases with the increase of injection dose. The immune dose of protein antigens is wider than that of polysaccharide antigens. Generally speaking, the initial immunization dose for mice is 50 μ g to 400 μ g/time, for rats it is 100 μ g to 1000 μ g/time, and for rabbits it is 200 μ g to 1000 μ g/time twice. The strengthening dose is 1/5-2/5 of the first dose.
The immune dose is related to the injection route. Generally speaking, intravenous injection has a higher dose than subcutaneous injection, and subcutaneous injection has a higher dose than palmar and plantar subcutaneous injection. Lymph node injection can also be used. The injection dose with adjuvant is smaller than that without adjuvant. For rabbits, using Freund's complete adjuvant requires injection of 0.5mg to 1mg/kg per time. If Freund's incomplete adjuvant is used, the injection dose should be more than 10 times larger. To prepare highly specific anti serum, low-dose antigen short-range immunization method can be used. If high titer anti serum is needed, high-dose long-range immunization should be used. The immune cycle is prolonged and can occur in small amounts multiple times. For those with a short immune cycle, they should have a large number of episodes. The interval between two injections should be of appropriate length. If it is too short, it will not have the effect of a second reaction. If it is too long, it will lose the sensitivity of the previous stimulation. The general interval should be 5-7 days, and for those with adjuvants, it should be around 2 weeks. If the purity of injected Ig is high, it is generally not easy to cause allergic reactions. For example, injecting serum, even in small amounts, can easily cause allergic reactions when re immunized, so measures must be taken.
7、 Antibody identification
Whether used for diagnosis or treatment, the purpose of preparing antibodies is to require a high titer for antibody potency identification. The required potency of antibodies prepared from different antigens varies. There are many methods for identifying potency, including test tube agglutination reaction, agar diffusion test, enzyme-linked immunosorbent assay (ELISA), etc. The antibodies prepared from commonly used antigens generally have a method for determining their potency for comparison. The potency of anti antibodies is generally determined by agar diffusion test.
The specificity of antibodies refers to their ability to recognize corresponding antigens or similar antigenic substances. The high specificity of antibodies results in strong recognition ability. The specificity is usually measured by the cross reactivity rate. The cross reactivity rate can be determined by competitive inhibition test. Perform competitive inhibition curves with different concentrations of antigens and approximate antigens, calculate their respective binding rates, determine their concentrations at IC50, and calculate the cross reactivity rate according to the following formula. If the IC50 concentration of the antigen used is pg/tube, and the IC50 concentration of some similar antigen substances is almost infinite, it indicates that the cross reactivity rate of this serum with other antigen substances is approximately 0, indicating that the specificity of the serum is good.
The affinity of antibodies refers to the degree of firm binding between antibodies and antigens. The level of affinity is determined by the size of the antigen molecule, the appropriate stereoconfiguration between the binding site of the antibody molecule and the antigenic determinant clusteThe intermolecular forces that help maintain the stability of antigen antibody complexes include hydrogen bonding, hydrophobic bonding, Coulomb force, van der Waals force, and spatial repulsion of genes with opposite side chain charges. Affinity is often represented by the affinity constant K, which is measured in L/mol. Typically, K ranges from 108 to 1010/mol, with some even reaching up to 1014/mol. The determination of antibody affinity is of great significance for screening antibodies, determining the purpose of antibodies, and verifying the uniformity of antibodies.
8、Anti serum cryopreservation
After harvesting the anti serum, add 1/10000 thiomersal or 1/10000 sodium azide for preservation, or add an equal amount of neutral glycerol, package in small bottles, and store at a low temperature below -20 ℃. The antibody potency remains unchanged for several months to years. Be careful to avoid repeated freezing and thawing. Anti blood therapy can also be used
Store after freeze-drying.
9、Antibody preparation
The preparation of polyclonal antibodies generally includes the following steps:
1、Prepare antigens.
2、Select experimental animals.
3、Animal immunity.
4、Take blood samples for testing to see if immunization is successful.
5、If successfully immunized, kill the experimental animals and collect all serum.
6、Purify the antibody.
7、Identify antibodies. Including purity and specificity.
10、Immune methods
You can use one of the following methods for immunization.
(1) Lymph node injection method: ① Subcutaneous (or intradermal) injection of 50mg live BCG vaccine (approximately 0.30ml per side) into the plantar area of the rabbit's hind feet. After 7-10 days, the lymph nodes in the rabbit's plantar and hamstring muscles swell; ② Inject 0.50ml of IgG emulsified antigen with complete adjuvant (containing IgG 5mg/ml, penicillin 1000U/ml, streptomycin 1000 μ g/ml) into the swollen lymph nodes on both sides; ③ If necessary, repeat step ② once after 14 days; ④ After 7 days, inject 0.50ml of IgG emulsified antigen with complete adjuvant (containing IgG5mg/ml, penicillin 1000U/ml, streptomycin 1000 μ g/ml) into each lymph node on both sides; ⑤ 5-7 days later, blood will be collected from the ear vein. Measure serum potency.
(2) Subcutaneous multi-point injection method: ① Inject 0.10ml of complete adjuvant antigen (IgG content 5mg/ml) into the palms of both sides of the rabbit (plantar injection); ② 7-10 days later, subcutaneous injection of antigen containing incomplete adjuvant 5 at multiple points on both sides of the spine (2 points each on the neck, chest, and lumbar spine, a total of 6 points), with a dose of 0.50ml per point; ③ 7-10 days later, repeated injection on both sides of the spine; ④ Blood test will be conducted 7-10 days later. Repeat step ③ for those who do not meet the requirements.
(3) Multi pathway combined injection method: ① Subcutaneous injection of 0.50ml of complete adjuvant antigen (IgG dose of 5mg/ml) into the inner side of the palms (soles) on both sides; ② After 14 days, multiple subcutaneous injections containing incomplete adjuvant antigens were administered; ③ After 7 days, 2ml of antigen without adjuvant was injected into the ear vein; ④ Serum antibody titers were measured, and steps ③ were repeated for those who did not meet the standards, with appropriate increases in IgG levels.
11、 Potency determination
Adopt agar diffusion method.
⑴ Dilute the serum ratio and add it to the peripheral well.
⑵ Add animal Ig to the middle hole.
⑶ Wet box expansion at 37 ℃ for 24 hours, observe the results.
12、 Result judgment
1．The potency determination of anti Ig is qualified if the agar diffusion potency reaches 1:16 or 1:32.
2．The purity identification adopts the Qiong expansion method. Add anti Ig serum to the middle well, and Ig and standard Ig to the peripheral well. The molecular structure on the anti Ig and I antigen that can cause the body to produce antibodies is called the antigenic determinant. There can be several different antigenic determinants on an antigen, which leads to the production of several different antibodies in the body, ultimately resulting in plasma cells producing antibodies. A plasma cell population that only acts on one antigenic determinant is a pure line, which is called Clone in English. The specific antibody produced by a clone is called a monoclonal antibody. Monoclonal antibodies can bind specifically to a single antigenic determinant, just like a missile hitting its target with precision. On the other hand, even within the same antigenic determinant cluster, antibodies can be produced by several clones in the body, forming a mixture of several monoclonal antibodies, known as polyclonal antibodies.
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