Methods Immunization

1. An efficient immunization protocol resulting in the availability of high numbers of highly proliferating antigen-secreting B-cells is one of the most significant factors in generating successful hybridomas.

2. Complete Freund's adjuvant (0.2-0.5 mL) or Ribi Adjuvant System (0.1 mL), at 1:1 mixtures with antigenic material, are used as i.p. injections for initial immunization to boost the immune response. Incomplete Freund's adjuvant (1:1) is used in subsequent immunizations via s.c., i.m., or i.p. administration.

3. Immunization protocols vary widely, depending on the investigator's experimental objectives, model, and personal style. Typically, 50-100 p,g of protein or 106-107 cells mixed with the adjuvant of choice are used for initial immunization, followed by booster injections containing 10-50 p,g of protein or 105-106 cells in 1:1 ration with incomplete adjuvant. To avoid neutralization of the injected antigen by circulating antibodies, the following immunization schedule is most commonly utilized:

Day 0—primary immunization, i.p.

Day 36—serum collection and titration

Day 59—serum collection, harvest of splenocytes, cell fusion

4. Blood for subsequent serum titration is collected (not to exceed 0.5 mL) from the retro-orbital sinus under IACUC-approved anesthesia guidelines. The timeline for the fusion is determined based on the most recent serum titration data obtained using the screening method of the investigator's choice.

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