Before starting
Discussion of project: choice of antigen for immunization and for screening.
Unit 1: Immune response
- Take preimmune sample from each mouse.
- Inject mice with antigen in CFA.
- Boost with antigen in IFA.
- Take test bleeds from all mice.
- Screen serum for response to antigen by ELISA.
- Other screening tests (i.e., Western blot, immunofluorescent staining) can be done in collaboration with client.
- If titers are satisfactory then continue to fusion step.
- Otherwise, do a boost injection, second test bleed, and second screening.
- If immune response is still poor, re-evaluate project (i.e., change strain of mice and/or antigen).
Unit 2: Successful Fusion
- Boost selected mouse, perform fusion three days later.
- Screen clones by ELISA (other screens can be done in collaboration with client).
- Select up to 5 wells for cloning (freeze down other possibly interesting clones).
- If fusion is unsuccessful (no ELISA-positive clones) a repeat fusion will be performed at no extra charge.
Unit 3: Cloning /expansion
- Clone by limiting dilution.
- Grow up first subclones.
- Screen by ELISA (other screens can be done in collaboration with client).
- Freeze 1st subclones.
- Do second round of cloning.
- Screen for positives as before.
- Freeze down 2nd subclones (three per original subclone well).
- Grow up 100ml of tissue culture supernatant from each selected monoclonal.
- Cells (frozen or in culture, as desired) and 100 ml tissue culture supernatant from each final clone will be delivered to user.