Antibodies, as a research tool, play a crucial role in current scientific research and medical work, thus the demand for antibody preparation is increasing. In recent years, there has been an increasing amount of research on the classification of antibodies, antigen preparation, immunization of animals, and selection of immunization methods in the process of antibody preparation.
1 Classification of antibodies
Antibodies are divided into four categories: natural antibodies, monoclonal antibodies, polyclonal antibodies, and genetically engineered antibodies.
In 1975, Dr. Milshuta from the University of Cambridge in the UK fused lymphocytes and myeloma cells to produce hybridoma cells. Hybridoma cells overcame the difficulty of lymphocyte cloning and were able to infinitely proliferate lymphocytes in vitro. This infinite proliferation ability is the transplantation of myeloma cell proliferation ability. When lymphocytes with unlimited proliferation ability are stimulated with cancer cells as antigens, they will continuously synthesize antibodies with single immune function. This type of antibody produced by a B lymphocyte receiving an antigen determined cluster that stimulates the body is called a monoclonal antibody, which has high specificity, selectivity, and specificity.
In 1888, Emile and Yersin discovered that diphtheria bacteria could produce exotoxins. Subsequently, in 1890, Behring and Kitasato immunized animals with diphtheria exotoxin and discovered a substance in the animal's serum that could neutralize diphtheria exotoxin, called an antitoxin. They then successfully cured a girl with diphtheria disease using the same method by immunizing the serum. Stimulating the body with multiple antigenic determinants like this. A series of antibody producing cells will bind to antigens to varying degrees, producing different types of monoclonal antibodies in the blood. These monoclonal antibodies, which are mixed together and stimulated by one antigen, are called polyclonal antibodies.
1.1 Comparison between monoclonal antibodies and polyclonal antibodies
Monoclonal antibodies and polyclonal antibodies each have their own advantages and disadvantages. Overall, monoclonal antibodies have high specificity and can continue to produce identical antibodies after successful preparation. However, monoclonal antibodies cannot undergo precipitation and agglutination reactions, so many detection methods cannot be completed using monoclonal antibodies. Moreover, the reaction intensity is not as strong as that of polyclonal antibodies, and the preparation technology is complex, time-consuming, labor-intensive, the experimental cycle is long, and the price is high. Therefore, polyclonal antibodies are commonly used in the field of scientific research.
1.2 Preparation principle and detection method of polyclonal antibodies
When the antigen is injected into the experimental animal, it stimulates the reticuloendothelial cell system, causing a large proliferation of lymphocytes in lymph nodes and spleen. About 7 days after the first injection, antibodies can be observed in the serum, but the concentration of antibodies remains at a low level, and the titer of antibodies will reach its maximum value after about 10 days. However, the results of the secondary immune response generated by injection of the same antigen are significantly different, with a significantly increased synthesis rate and longer retention time of antibodies compared to the primary immune response. Furthermore, the titer of the antibody was detected by Elisa method, and the specificity of the antibody was detected by Western blot method.
2 Antigen acquisition method
Prokaryotic expression and peptide synthesis of target proteins each have their own advantages and disadvantages. Prokaryotic expression can obtain gene expression products in a relatively short period of time and at a lower cost, but there are also some exogenous genes that cannot be expressed in prokaryotic form. Peptide synthesis is the solid-phase or liquid-phase synthesis of analyzed peptide antigens, which has high antigenicity but also high cost. Therefore, in the actual experimental process, prokaryotic expression and peptide synthesis should be effectively combined to complete the preparation of polyclonal antibodies.
2.1 Prokaryotic expression system
Among different types of expression systems, the prokaryotic expression system was the earliest adopted. This technology mainly involves transforming prokaryotic expression vectors containing cloned target gene fragments into prokaryotic expression strains (usually Escherichia coli), and obtaining the desired protein under IPTG induction and purification. The required time is relatively short and the cost is low. Moreover, Escherichia coli has a clear genetic background, short cycle, safe use, and easy operation, making it the preferred expression system for exogenous genes.
2.1.1 Prokaryotic expression vector
The vectors used in genetic engineering are divided into cloning vectors and expression vectors. Cloning vectors can replicate and amplify exogenous genes in recipient cells; And expression vectors are suitable for expressing exogenous genes in recipient cells. Prokaryotic expression vectors typically exist in plasmid form, and a typical expression vector should have a promoter, replicon, screening marker, multiple cloning sites, fusion tag (if any), and stop codon. When constructing prokaryotic expression vectors, it is important to learn to look at plasmid profiles and first determine the position of the promoter (Ori); Next, we need to look at the filtering tags; Again, we need to look at multiple cloning sites; Finally, determine whether there are promoters and terminators on the plasmid.
Classification of prokaryotic expression vectors: When prokaryotic cells express exogenous genes, they can be classified into fusion expression and non fusion expression based on their expression types. Foreign proteins expressed through non fusion do not fuse with bacterial proteins or peptides, and usually exist in the form of inclusion bodies. Common prokaryotic expression vectors include the non fusion expression vector pKK223-3, which has a strong tac (trp lac) promoter; Fusion expression vector pGEX; Secretory cloning expression vector piNlll system, fusion protein vector PET system, etc.
The pET system is currently the most widely used expression vector, and the target gene is cloned into the pET plasmid vector. When there is T7 RNA polymerase and T7 phage promoter in the cell, the transcription of the host's own gene cannot match the transcription system of the T7 phage promoter. After induction of expression, the exogenous target protein usually accounts for more than 50% of the total cell protein within a few hours. However, Escherichia coli itself does not express T7RNA polymerase, so it is necessary to introduce exogenous TTRNA polymerase into the host bacteria. Without the addition of inducers, the exogenous gene remains silent and transcription does not occur; By adding inducers to control the production of T7RNA polymerase, the expression level of exogenous protein products can be regulated.
2.1.2 Selection of host bacteria
The selection of host strain for expression is also a factor that must be considered in the process of prokaryotic protein expression. The increase of endogenous protease in bacterial strains can cause instability of exogenous expression products, so protease deficient strains such as B1221 are often used in prokaryotic expression. B121 (DE3) lysogenic bacteria added T7RNA polymerase gene, which contains downstream T7RNA polymerase gene of lacUV5 promoter and lac I inhibitory gene. T7RNA polymerase was introduced into the host through lysogenic bacteria. After the constructed expression vector was transformed into the expression strain, expression was induced by isopropyl-p-D thiogalactoside (IPTG) under lac induced regulation.
2.1.3 Factors affecting prokaryotic expression of exogenous genes
The factors that affect the expression of exogenous genes in the E. coli system include: ① Selection of expression vector: Insertion of auxiliary gene sequences into the expression vector to form fusion protein expression. The expression of fusion signal peptides (PelB, OmpA, MalE, PhoA, etc.) can be secreted into the cytoplasm or extracellular space through the Sec pathway, which is conducive to the formation of disulfide bonds and avoids the hydrolysis of cytoplasmic proteases and the extension of N-terminal methionine. Research has shown that the dual crystal amino acid transport system (Tat) can effectively secrete recombinant proteins that fold correctly. Common purification labels include 6-His, GST, CAT, etc. When using streptavidin binding peptide (SBP) and Ca2+- dependent calmodulin binding peptide (CBP) as purification labels, removing label dependent small molecules from the eluent can achieve mild and specific elution; ② The use of codons in exogenous genes, if they contain a considerable number of rare codons, will result in lower expression efficiency in prokaryotes; ③ MRNA stability impact; ④ Control of cultivation conditions.
2.2 Peptide synthesis method
Peptides are a type of compound with a molecular structure between amino acids and proteins, formed by the binding of one or more amino acids in a certain order through peptides. The main synthetic pathways include chemical synthesis and biosynthesis. The chemical synthesis of peptides can be divided into liquid-phase synthesis and solid-phase synthesis. Peptide chemical synthesis classification: Liquid phase segmented synthesis is a method of synthesizing peptides spontaneously in solution based on the chemical selectivity or specificity of peptide fragments. The commonly used connection techniques include: natural chemical connection, photosensitive auxiliary group connection, chemical region selection connection, Staudinger connection, removable auxiliary group connection, and orthogonal chemical connection. The basic principle of solid-phase synthesis is to covalently bond the hydroxyl groups of the hydroxyl terminal amino acids of the peptide chain to an insoluble polymer resin, and then use the amino acids bound to the solid-phase carrier as the amino component. After removing the amino protecting group and reacting with excess activated carboxyl components, the peptide chain is extended. The solid-phase synthesis method simplifies and accelerates the multi-step synthesis, avoiding losses caused by manual operation and repeated material transfer.
3 Selection of animals used
The final step in obtaining polyclonal antibodies is animal immunization. Choosing appropriate immune animals is particularly important. Generally speaking, the selected protein antigen donor should not be too close to the immune animal strain, as being too closely related may not produce good antibodies, and may not even produce antibodies (such as rabbits and rats, chickens and ducks). Immune animals include small experimental animals such as rabbits, rodents, chickens, as well as large livestock such as sheep, horses, and goats. Among them, rabbits are the most suitable animals for preparing antibodies; Mice are generally used for monoclonal antibody preparation, and when a large amount of serum is required, large livestock are mainly used. The selection of animal gender, age, and quantity generally involves one or more female or young adult individuals. Being sick, infected, or hungry can all affect immune function, so healthy and strong animals should be selected. In the actual immunization process, animals need to be selected for immunization based on the different properties of immunogens. For protein antigens, most animals are suitable, with rabbits and goats being the most commonly used. When certain animals have similar substances present in their bodies, the protein antigen immune prototype deteriorates, such as insulin being less likely to produce antibodies in rabbits and IgE being less likely to produce antibodies in sheep.
4 Immune methods
4.1 Immune adjuvant
Freund's complete adjuvant and Freund's incomplete adjuvant are required during the immune process. Freund's incomplete adjuvant is generally made by mixing liquid paraffin with lanolin, and adding live BCG or dead Mycobacterium tuberculosis to the incomplete adjuvant becomes Freund's complete adjuvant. Due to the fact that adjuvants are oily substances, it is necessary to thoroughly mix Freund's adjuvant and antigen into an emulsion. The method for identifying the mixture is to drop a drop of emulsion into water. If the emulsion does not disperse and floats on the liquid surface, it indicates complete emulsification.
4.2 Immune dose and interval time
When immunizing animals, the individual state of the animal, the strength of antigenicity, and the size of antigen molecules should be considered when selecting the appropriate immunization dose. Generally speaking, after the first injection, the interval between each injection is about one week, with four injections. Before collecting immune serum, the antibody potency must be measured first, and blood collection can be carried out when it meets the requirements.
4.3 Immune pathway
The immune pathways include subcutaneous, intravenous or intradermal injection, intramuscular injection, intraperitoneal injection, lymph node injection, etc. Studies on chickens have shown that injecting into the leg muscles results in higher antibody titers; Studying the injection of Houhai acupoint on cows can also achieve higher efficacy; Rabbits and mice generally achieve better results when injected into the abdominal skin.
4.4 Collection of immune serum
After measuring the antibody potency, immune serum was collected and the animals were fasted for 24 hours before collection to prevent high blood lipids. At present, blood collection mainly includes venous blood collection method, carotid artery blood collection method, and cardiac blood collection method. Venous blood collection method can keep animals in a live state and can collect blood multiple times, but the disadvantage is that the amount of blood obtained each time is relatively small. The carotid artery blood collection method and cardiac blood collection method obtain a large amount of blood, but require proficient surgical techniques.
4.5 Preservation of immune serum
Before freezing the serum, divide it into several small bottles and take out one bottle each time. Avoid repeated freezing and thawing of the immune serum, which may cause a decrease in antibody potency.
The acquisition of polyclonal antibodies requires the acquisition of antigens, selection of immunized animals, immunization methods, and establishment of immune serum collection methods. In terms of antigen acquisition, exogenous gene sequences should be analyzed to select prokaryotic expression or peptide synthesis peptide fragments, and then suitable immune animals and immune sites should be selected based on the immunogen, ultimately obtaining high titer immune serum, providing essential experimental materials for scientific research and medical fields.
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