Cold Spring Harb Protoc. 2020 Mar 2;2020(3):100297. doi: 10.1101/pdb.prot100297.
Mice, rats, or hamsters are immunized by giving biweekly injections of a purified antigen, cultured cells, or cDNA. For mice, if a pure, soluble protein antigen is being used and is abundant, a dose of 50-100 µg in adjuvant at each immunization is a sensible general recommendation; for rats and hamsters, a dose of 100-200 µg is sufficient. Lower doses can be used for antigens with higher immunogenicity. Adjuvants (Freund's, Ribi, Hunter's TiterMax, ImmunEasy, or Alum) should be mixed with the immunizing antigen for the first two immunizations only; Complete Freund's adjuvant is only used with the first immunization. Subsequent immunizations are performed in phosphate-buffered saline (PBS) or normal saline, with or without Incomplete Freund's adjuvant. The choice of adjuvant is dependent on the subclass of immunoglobulin required. Over the course of the 6-wk immunization schedule, each animal usually receives a total of six injections (three subcutaneous and three intraperitoneal). Once a good titer has developed against the antigen of interest, regular boosts and bleeds are performed to collect the maximum amount of serum. For rats and hamsters, boosts should be spaced every 2-3 wk, and serum samples of 400-500 µL should be collected 10-12 d after each boost. For mice, boosts should be spaced every 2-3 wk, and serum samples of 200-300 µL should be collected 10-12 d after each boost.
小鼠、大鼠或仓鼠通过每两周注射一次纯化抗原、培养细胞或 cDNA 进行免疫。对于小鼠，如果使用的是纯可溶性蛋白抗原且含量丰富，每次免疫时在佐剂中的剂量为 50-100 µg 是合理的一般建议；对于大鼠和仓鼠，剂量为 100-200 µg 即可。对于免疫原性更高的抗原，可以使用较低的剂量。佐剂（弗氏佐剂、Ribi、Hunter 的 TiterMax、Immuneasy 或 Alum）仅应与前两次免疫中的免疫原混合使用；只有第一次免疫时才使用完全弗氏佐剂。随后的免疫在磷酸盐缓冲盐水 (PBS) 或生理盐水进行，有或没有不完全弗氏佐剂。佐剂的选择取决于所需的免疫球蛋白亚类。在 6 周的免疫计划过程中，每只动物通常总共接受六次注射（三次皮下和三次腹腔内）。一旦针对感兴趣的抗原产生了良好的效价，就会定期进行加强免疫和采血，以收集最大量的血清。对于大鼠和仓鼠，加强免疫应每 2-3 周进行一次，每次加强免疫后 10-12 天应采集 400-500 µL 的血清样本。对于小鼠，加强免疫应每 2-3 周进行一次，每次加强免疫后 10-12 天应采集 200-300 µL 的血清样本。
