| Animal |
5 Balb/c mice or 3 rats |
| Adjuvant |
Complete Freund's Adjuvant (CFA) initially, followed by incomplete Freund's Adjuvant (IFA) or T-MaxTM adjuvant for subsequent injections except final boost. |
| Immunogen |
Typically, proteins and conjugated peptides. The amount of immunogen used for each immunization is 10-100 ug per mouse/rat per injection. |
| Immunization |
The immunogen is diluted with sterile saline and combined with the appropriate adjuvant. The antigen and adjuvant are mixed thoroughly to form a stable emulsion, which is injected intraperitonally, providing enhanced immune response from the sustained presence of the immunogen. |
| Schedule |
| Day 1 |
Bleed 0.2 ml (yields 0.1 ml pre-immune serum)
1st immunization with antigen in CFA |
| Day 14 |
2nd immunization with antigen in IFA or T-MaxTM adjuvant |
| Day 21 |
Test bleed and ELISA |
| Day 35 |
3rd immunization with antigen in IFA or T-MaxTM adjuvant |
| Day 42 |
Test bleed and ELISA |
| Day 56 |
4th immunization with antigen in PBS or saline |
| Day 60 |
Cell fusion |
|
| Fusion |
Splenocyte fusions are performed on the mouse/rat shown to have responded the best to the immunization. |
| Expansion and Screening |
The fused cells are plated into 96-well culture plates and screened by ELISA to detect the positive clones. |
| Subcloning, expansion and cryopreservation |
The selected clones are then subcloned by limiting dilution until they are monoclonal and stable hybridomas. Two subclones from each parental clone are expanded into culture flasks, and 4-6 vials of cells for each subclonal cell line are cryopreserved. |
| Delivery |
The typical delivery includes two vials of frozen cells or one flask of live cell culture per clone and 5 ml of culture supernatant per clone. |
