General Protocol of Monoclonal Antibody Production:

1) Immunization animal: Select one healthy Balb/c mouse.

2) Adjuvants: Start with Complete Freund’s Adjuvant (CFA, Sigma), followed by Incomplete Freund’s Adjuvant (IFA, Sigma).

3) Antigen: Protein or KLH-conjugated peptide. Use 50–100μg of antigen for each immunization.

4) Immunization: Dilute the antigen with PBS and then mix it 1:1 with the corresponding adjuvant. Thoroughly mix the antigen and adjuvant to form a stable emulsion, draw the antigen mixture with a syringe, and perform multiple subcutaneous injections under the mouse’s skin. Inject 0.05–0.1ml per injection site. In this way, the antigen persists, enhancing the immune response until the required titer is achieved.

5) Day 0: Collect negative serum from the orbital venous plexus, 50ul per mouse; Day 1 Immunization 1: Immunize the mouse with 120μg of antigen mixed with Complete Freund’s Adjuvant (CFA); Day 14 Immunization 2: Immunize the mouse with 60μg of antigen mixed with Incomplete Freund’s Adjuvant (IFA); Day 28 Immunization 3: Immunize the mouse with 60μg of antigen mixed with Incomplete Freund’s Adjuvant (IFA); Day 35 Collection 1: Serum test ELISA, if OD value at 1:4000 is greater than 1.0, it’s positive, boost the mouse three days before fusion; if below 1.0, continue immunization and blood collection; Day 41 Collection 2; Day 42 Immunization 4, immunization method and dosage same as Immunization 3. Blood is collected once a week, immunization is done every two weeks. When serum test results are positive, proceed to fusion. Boost the mouse three days before fusion, subcutaneously immunize the mouse with 60μg of antigen dissolved in PBS.

6) Hybridoma fusion and screening: Three days after the boost, remove the mouse’s eyeball to collect positive serum, and fuse spleen cells with myeloma cells F0 using PEG. Seven days after cultivation, screen the supernatant of the fused hybridoma clones with the antigen to check their specificity and sensitivity; typically, initial screening and rescreening are required. Clones positive in ELISA can be further tested with WB, and selected clones must be subcloned at least two more times to obtain stable antibody-secreting monoclonals.

7) Large-scale antibody production: Positive clones selected for large-scale supernatant culture or ascites production (5 mice per clone). Purify the culture supernatant or ascites with Protein A/G to obtain purified antibodies, with a purity of more than 90%. On average, 3–5mg of antibody can be obtained per clone.