1. Obtain 5-10 knock-out mice (16), approx 2 mo in age (see Note 1).
2. The underside of the tail is vaso-dialated by applying extract of wintergreen topically or by heating with a lamp.
3. Nick the underside of the tail with a razor blade. Have a blood collection tube ready to catch the drops of blood (50-100 |L). Tape a piece of gauze over the cut to stop the bleeding, which is removed after 1 d.
4. Sera is prepared from the blood as described (13). First incubate the blood at 37°C for 1 h with occasional flicking to dislodge the blood clot. The tube is then transferred to 4°C for 2-16 h and spun at 10,000g for 10 min at 4°C. The serum is removed from the pellet and spun a second time for 10 min and stored at -20°C.
3.1.2. Immunization with the Antigen
1. Warm the adjuvant (TiterMax®) to room temperature and then vortex for 30 s.
2. Dissolve the immunizing protein in PBS.
3. Prepare an emulsion of adjuvant and antigen by first loading a syringe, which is all-plastic, through an 18 gauge needle with 0.5 mL of adjuvant.
4. Load a second syringe with half of the total antigen volume: in this case, 0.25 mL.
5. Remove the needles and connect the two syringes by an 18-gauge double-hub emulsifying needle.
6. Push the antigen into the adjuvant first, mixing the adjuvant with the antigen by forcing the solution back and forth through the needle for approx 1 min. It is important to push the antigen into the adjuvant first so that the aqueous phase enters the oil phase.
7. After 1 min, take the syringes apart and load the empty syringe with the remaining 0.25 mL of antigen. Reconnect and emulsify for another min. It will become difficult to continue mixing when the antigen-adjuvant is properly prepared, as it has the consistency of whipped cream (see Note 2).
8. Load the antigen/adjuvant mixture into a 28-gauge hypodermic needle and deliver 25 |L into two sites subcutaneously at the base of the tail (see Note 3).
9. The yield of antigen/adjuvant will be approx 30%, thus, this protocol gives enough to immunize 6 mice.
10. Fourteen d later, repeat steps 1-10.
11. Twenty-one d later, collect serum from each animal and assay for the presence of antibodies to the antigen of interest. Use the same assay that will be used to screen the hybridomas by ELISA.
12. Three to four wk after the last boost but 5 d before harvesting the spleen, inject the animal previously identified as making antibody intraperitonealy with 20-100 |g of soluble antigen (no adjuvant) (see Note 4). On the second d, immunize the animals intravenously with soluble antigen (see Note 5). On the fifth d, harvest the splenocytes for fusion.
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