If recombinant protein is used to immunize mice for polyclonal antibody production and only 500 μg of protein is available, how should the adjuvant and immunization doses be arranged?

When emulsifying, it is essential to confirm a water-in-oil state. Mix the protein solution and CFA or IFA at a 1:1 volume ratio, connect two glass or plastic syringes, and push back and forth through a three-way valve, operating on ice. Push until resistance is obvious; a drop placed on the surface of cold water that does not disperse and appears as a milky white droplet is ready. If the protein volume is too small, first dilute with normal saline or PBS to an appropriate volume before emulsifying; do not use solutions containing high concentrations of salt or detergents. Use within 1 hour after emulsification; do not keep any remainder.

If there are concerns about weak immunogenicity, CFA/IFA can be selected as adjuvants; these classic adjuvants remain relatively potent for polyclonal antibody production. Alternatively, perform a small-scale test by mixing antigen with adjuvant first—do not use up everything at once. Impurity proteins within the 90% purity preparation may also be immunized, potentially resulting in contaminating antibodies in the subsequent polyclonal antisera; if high specificity is required downstream, antigen affinity column purification can be performed after blood collection. An immunization dose in the 20–50 μg range is usually sufficient; the key lies in emulsion quality and booster interval.

For the injection route, I recommend subcutaneous multi-site administration rather than intraperitoneal. Subcutaneously, 4–6 sites can be used per animal along the back and both flanks, with about 50 μL per site, which can enhance antigen presentation and reduce local ulceration. Although intraperitoneal injection is faster to perform, it does not necessarily produce a better antibody response to soluble antigens, and the animals' condition may be affected after intraperitoneal injection of oil adjuvant. If the protein immunogen is weakly immunogenic, CFA can be used for the first immunization, followed by IFA, rather than using CFA every time.

500 μg at 90% purity is enough for 4 Balb/c mice, but you need to budget carefully. For 4 mice, it is recommended: prime each mouse with 30–50 μg emulsified with an equal volume of CFA; on days 14 and 28, boost each with 15–25 μg plus IFA. For 4 mice, the prime totals at most 200 μg, the two boosts total about 160 μg, and even with emulsification loss, there is still enough left for coating and titer detection. If you want to use 5 mice, reduce the prime to 20–30 μg and the boosts to 10–20 μg. Do not inject all the protein at once; small doses with multiple boosts are more favorable for antibody affinity.

Don't wait until the end to measure the titer. About 10 days after the second immunization, collect blood via tail vein, use serum or plasma for ELISA, coat with 0.5–1 μg/mL original antigen, and test serial dilutions of the secondary antibody. If the titer is only a few thousand, you can add a third immunization or switch to a small amount of aqueous phase for boosting. Before the final blood collection, fasting is recommended; cardiac puncture or orbital bleeding can yield 0.4–0.7 mL of blood at one time. Of the 500 μg protein, reserve 30–50 μg for coating the ELISA plate; don't inject it all into the mouse.