Modified Immunization Protocol for Anamnestic Antigens

When it is known from other work that the antigen is highly glycosylated or comes from a source known to be rich in polysac-charides (bacterial cell walls) it is highly unlikely that the animal will ever produce a classic full immune response. Usually these antigens do not produce quiescent B-cells after the rest period and each immunization is seen as a primary challenge. Substances that invoke this incomplete response are known as anamnestic antigens, and immunoglobulins produced to them are always class M. There is no point in carrying out a full immunization protocol over several months, and a shortened one is recommended.

1. Mix 0.15 mL of 0.5 mg/mL solution of antigen with 0.15 appropriate adjuvant and mix (volumes based on group of three mice).

2. Inject 0.1 mL of adjuvant/antigen mixture per dose IP. Repeat on d 14.

3. Take a test bleed by tail tip amputation under light anesthesia on d 21 and assess for circulating antibody. All antibody produced will be IgM and so assessment method (ELISA) must take this into account. Mark the mice by ear punch or tattoo for subsequent identification.

4. Give the mouse exhibiting the highest titre of circulating antibody an IP injection of 0.1 mL (0.5 mg/mL solution) of antigen on d 23.

5. Harvest the spleen and perform the cell fusion on d 30.

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