STAGE I: Immunization

• Ten virus antibody-free mice will be immunized with the customer's antigen. The mice will be housed in an isolator during the immunization period. We immunize ten mice to increase our chance of an immunogenic reaction

• In order to get a better antibody response, the antigen is prepared with an adjuvant such as FCA (Freund's complete adjuvant) for the primary immunization

• The emulsion contains heat-killed mycobacterium that ensures the antigen is released slowly into the animal's circulatory system

• The bacteria in the adjuvant stimulates the animal’s immune system

• Additional boosts or immunizations are necessary for high antibody levels

  FIA (Freund's incomplete adjuvant) is used

• Common routes for injection are i.p., i.m., i.v., s.c.

• For hybridoma development, s.q. is typically used. five-to-100 ug of antigen are used for each immunization. We ask the client for two-three mg of antigen

• At six weeks, following the primary immunization and the first boost, the mice will be tested for antibody titer. The antibody will be determined by ELISA assay unless the customer has an alternative assay.

• If additional boosts are required, they will be administered and the mice will be retested for antibody titer

  Once immunization is complete, the mice will be held for three-to-four weeks before fusion

• Three-to-five days before the fusion, a mouse will be selected for the pre-fusion boost

STAGE II

1) Fusion

• The spleen from the mouse will be harvested and all spleen cells fused with a myeloma cell line. We generally use NS-1 for the fusion
• Generally six-to-ten 96-well panels will be plated
• Cells will be grown in HAT (hypoxanthine, aminopterin and thymidine) selection medium. This selectively kills cells deficient in the enzyme (HGPRTase), which is essential for the operation of the salvage pathway for making nucleic acids. The myeloma and spleen cells will die off
• A maximum of three separate fusions will be performed

2) Screen and select

• All 96 well panels will be screened by ELISA for antibody positive wells
• The standard production will be screened with a single ELISA antigen
• Additional ELISA antigens may be added as an option
• About 15 positive wells from the ELISA assay will be selected to be expanded and frozen for possible cloning
• Tissue culture supernatant (0.2 ml per clone) can be sent to the customer for further testing

STAGE III

1) First cloning

• A maximum of six positive wells will be selected for cloning
• They will be cloned by the limiting dilution method. In this method cells are diluted so that they can be individually pipetted into separate wells
• Isolated colonies will be tested by ELISA and positives selected to be expanded for the next cycle of cloning
• Colonies should be visible after ten-to-14 days
• Cells from these positive clones will be frozen as backup

2) Final cloning

• Positive clones from the first cloning cycle will be selected for the second cycle
• Isolated colonies will be tested by ELISA and positives selected to expand. Several cycles of cloning may be required in order to obtain true, stable clones. Cells from these positive clones will be frozen as backup

3) Selection or non-HT requirement

• A maximum of three positive clones will be further selected for clones, which grow in medium lacking HT

4) Frozen stock

• The final three clones selected will be expanded and a small stock of each will be frozen. A small amount of tissue culture supernatant (two-to-five ml) will be prepared for each clone. Each clone will be isotyped

NOTE: Our service allows for the development of up to three, five or ten positive clones.