Mechanism of Antibody Production in Animals: it involves the fusion of immune B cells from an immunized animal. Following fusion, the cells are screened to identify clones that produce antibodies specific to the target antigen. Antibody production begins with a strong immune response triggered by antigen stimulation in immunized animals, which relies on efficient animal immunization. In practice, the actual immunization effect is measured by detecting the antibody titer in the serum【1】.
Antigens in Animal Immunization
To prepare high-quality immune antibodies with strong specificity, high titer, and good affinity, suitable antigens are essential. The basic characteristics of antigens include immunogenicity and reactivity. Immunogenicity refers to the ability of an antigen to interact with antigen recognition receptors on T cells and B cells, inducing an immune response. Reactivity refers to the characteristic of an antigen binding specifically to its corresponding antibody, also known as immunoreactivity. The reactivity of an antigen depends on the epitopes, which are the regions where the antigen specifically binds to the antibody molecule. A single antigen molecule can have different epitopes【2】. The reactivity is closely related to the nature, spatial position, stereoconfiguration, and species differences of the epitopes. Immunogenicity is related to various factors of the antigen itself【3-6】, including:
1. Heterogeneity: Self or autologous antigens typically have poor immunogenicity. The greater the heterogeneity between the antigen and the host organism, the stronger the immune response.
2. Molecular Size: The smaller the antigen's molecular weight, the weaker its immunogenicity. For example, molecules with a molecular weight greater than 100,000 Da are usually active immunogens, whereas those smaller than 5,000-10,000 Da have poor immunogenicity.
3 .Chemical Nature and Composition: Antigens are primarily composed of proteins, polysaccharides, lipids, or nucleic acids. Proteins have the strongest immunogenicity, followed by polysaccharides. Lipids and nucleic acids generally do not become immunogens and often need to combine with proteins to activate an immune response. Generally, the more complex the chemical composition of a substance, the stronger its immunogenicity.
4.Degradability: Antigens that are easily phagocytosed usually have higher immunogenicity because, for most antigens (T-dependent antigens), the development of an immune response requires the antigen to be phagocytosed, processed, and presented to helper T cells by APCs.
Based on the immunogenicity and reactivity of antigens, they can be classified as complete antigens or incomplete antigens. Complete antigens possess both immunogenicity and reactivity【7】. Incomplete antigens only have reactivity and lack immunogenicity; they are also known as haptens【8】. Haptens alone do not induce an immune response but can acquire immunogenicity when combined with protein carriers to form hapten-carrier conjugates. For instance, synthetic peptide antigens are often too small to elicit a significant immune response from the host's immune system and need to be cross-linked with carrier proteins such as KLH, BSA, or OVA to become complete antigens capable of stimulating the host to produce the corresponding antibodies. It is important to note that such conjugates can stimulate the immune system to produce antibodies against both the hapten and the protein carrier, so another different carrier protein must be used for antibody screening.
To achieve the best immunization effects and obtain high-quality antigen-specific antibodies, the nature of the antigen, as well as the procedures and choice of immunization animals, are crucial.
Preparation and Emulsification of Antigens
Antigens for immunization are generally available in two forms: solution and lyophilized powder. Lyophilized powder should be fully dissolved in Saline or PBS at room temperature for 30 minutes before use. The antigen solution should be freshly prepared and diluted with sterile Saline or PBS. Adjuvants are substances co-injected with antigens into animals to non-specifically enhance the immune response to the antigen and are also known as non-specific immune enhancers. Soluble antigens are often immunized using adjuvants, forming stable emulsions for alpaca immunization. Particulate antigens are usually immunized without adjuvants, using PBS or saline to create a 1% suspension for immunization.
Common methods for emulsifying antigens include mortar grinding, syringe injection, ultrasonic emulsification, and mechanical stirring【9】. The most commonly used adjuvants for antigen immunization are Complete Freund's Adjuvant and Incomplete Freund's Adjuvant. The primary difference between them is that Complete Freund's Adjuvant contains BCG, with mineral oil and lanolin as main components, enhancing antigen immunogenicity and altering the body's immune response, thereby boosting immunity or increasing antibody production and prolonging antigen retention in the body. The degree of antigen emulsification directly affects the immunization effect, so quality checks must be conducted post-emulsification. When emulsified with Freund's adjuvant, the resulting emulsion should form an oil-in-water state. The quality check method involves dropping the emulsion onto the surface of cold tap water, where a qualified emulsion should maintain intact drops without dispersing (see Figure 1).