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).

The host animals generally chosen for the preparation of nanobodies are alpacas【10-12】. Factors such as the animal's genetic background and nutritional status directly influence the strength of the immune response. Some animals may have congenital immune deficiencies, leading to failed immunizations. A deficiency in vitamins and amino acids can reduce the immune function of the body. Poor environmental hygiene, including the presence of many pathogenic microorganisms in the pen and surrounding environment, can affect the immunization effect if animals are infected during the immunization period. The best candidates for immunization are young adult alpacas aged 2-3 years, as their immune systems are fully developed. It is particularly important to avoid using pregnant females for immunization.

Health Status of Alpacas Before Immunization: The alpaca should weigh approximately 50-70 kg and be around 2-3 years old. It should have a normal physical development, good nutritional status, and a bright coat. The alpaca should exhibit normal movement and behavior, without any signs of lameness or incoordination, and should be alert and responsive. If there are no visible external injuries upon examination, the alpaca can proceed with the immunization experiment (see Figure 2). Before immunization, a special tag should be fixed to the alpaca's neck, indicating its identification number. Blood should also be drawn to determine the presence of any naturally occurring cross-reactive antibodies. To ensure the health of the animals throughout the immunization process, the source and breeding environment of the alpacas should be controlled. Due to individual differences in immune response, multiple animals can be selected for immunization.

In practice, the combination of immunization dose, immunization site, and immunization interval has a significant impact on the immunization outcome. It is necessary to monitor the titer of the immune serum and make adjustment based on the actual immunization results.

1.Immunization Routes: Different immunization routes include subcutaneous injection in lymph node-dense areas and intravenous injection. Generally, the subcutaneous route is preferred over the intravenous route because intravenously injected antigens enter the spleen first, whereas subcutaneously injected antigens enter the local lymph nodes first. For alpacas, multiple injections are typically administered near the lymph nodes on both sides of the neck.

2.Immunogen Dose: The injection dose of the immunogen varies significantly with the nature of the antigen. Factors to consider include the antigen's immunogenicity, molecular weight, purity, the state of the individual animal, the immunization route and timing, and the use of adjuvants. A dose that is too low may not induce a sufficiently strong immune stimulus, while an excessively high dose may lead to immune tolerance. Within a certain range, antibody titer increases with the increase in injection dose. Generally, higher antigen amounts and longer intervals allow for larger doses. For pure soluble antigens, the initial immunization is typically mixed with an equal amount of Complete Freund's Adjuvant, with an immunization dose of 500 μg. Subsequent doses can be the same or half of the initial dose. For particulate antigens (cells), the immunization dose is 5*107 to 1*108 per injection. 
 
 3.Immunization Schedule: The schedule generally varies with the antigen, immunization method, and whether adjuvants are used. The interval can range from several days to weeks. The interval between the initial immunization and booster immunization is usually 2 weeks, with a total of 4 immunizations. Haptens require a longer immunization period to achieve high titers, and 1-2 additional immunizations may be needed. The immunization time and schedule can refer to Table 1 (Alpaca Immunization Schedule). 
 
 4.Determining Antibody Titer: Typically, blood can be collected 7-10 days after booster immunization (see Figure 4) to separate serum and determine antibody titer. For small molecules and polypeptide fragments with uncertain immunogenicity, blood should also be collected 14 days after the initial immunization to determine the titer and confirm the antigen's immunogenicity and the animal's reactivity. Common methods for determining antibody titer include ELISA and FACS.

[1] Newmark P. Nobel prize for Japanese immunologist. Nature, 1987, 329(6140): 570.

[2] Greenspan N S. Dimensions of antigen recognition and levels of immunological specificity[J]. 2001, 147-187.

[3] Schmitt M W, Prindle M J, Loeb L A. Implications of genetic heterogeneity in cancer[J]. Annals of the New York Academy of Sciences, 2012, 1267(1): 110-116.

[4] Schellekens H. Factors influencing the immunogenicity of therapeutic proteins[J]. Nephrology Dialysis Transplantation, 2005, 20(suppl_6): vi3-vi9.

[5] Ratanji K D, Derrick J P, Dearman R J, et al. Immunogenicity of therapeutic proteins: influence of aggregation[J]. Journal of immunotoxicology, 2014, 11(2): 99-109.

[6] Kharaziha M, Baidya A, Annabi N. Rational design of immunomodulatory hydrogels for chronic wound healing[J]. Advanced Materials, 2021, 33(39): 2100176.

[7] Murphy, Kenneth. Weaver, Casey. Janeway's immunobiology Ninth edition. New York, NY, USA: Garland Science, Taylor & Francis Group, LLC. 2017. ISBN 978-0-8153-4505-3. OCLC 933586700

[8] Landsteiner, Karl. The Specificity of Serological Reactions, 2nd Edition, revised. Courier Dover Publications. 1990. ISBN 0-486-66203-9.

[9] Lou Y F, Zhang X J, Liu J L, Wang R A, Liu S C, Meng M. Effects of different solvents on the polypeptide antigen’s emulsification[J]. Medical Reserch and Education, 2015, 32(3): 1-5.

[10] Salvador J P, Vilaplana L, Marco M P. Nanobody: outstanding features for diagnostic and therapeutic applications[J]. Analytical and bioanalytical chemistry, 2019, 411: 1703-1713.

[11] Beghein E, Gettemans J. Nanobody technology: a versatile toolkit for microscopic imaging, protein–protein interaction analysis, and protein function exploration[J]. Frontiers in immunology, 2017, 8: 276923.

[12] Bélanger K, Iqbal U, Tanha J, et al. Single-domain antibodies as therapeutic and imaging agents for the treatment of CNS diseases[J]. Antibodies, 2019, 8(2): 27.