| Animal |
10 Balb/c mice or 6 rats |
| Adjuvant |
Complete Freund's Adjuvant (CFA) initially, followed by incomplete Freund's Adjuvant (IFA) for all subsequent injections except final boost. |
| Immunogen |
10 mg or more purified antibody from customer. Digest the target antibody with pepsin to produce high-purity F(ab')2 fragment as immunogen for hybridoma screening. |
| 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 intraperitoneally, 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 |
| Day 21 |
Test bleed and ELISA |
| Day 28 |
3rd immunization with antigen in IFA |
| Day 35 |
Test bleed and ELISA |
| Day 42 |
4th immunization with antigen in saline or PBS |
| Day 47 |
Cell fusion |
|
| Fusion |
Test bleed antisera samples are tested by ELISA using the target antibody and a non-related negative antibody. Cell fusions are performed on the mice showing desired immune response to the immunogen. |
| Expansion and Screening |
The fused cells are plated into 96-well culture plates and screened by ELISA using the target antibody and a non-related negative antibody for the specific clone. The desired clones are then expanded into 24-well plates and isotyped. |
| Subcloning |
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. |
| Characterization |
Antibodies from the positive culture supernatants are tested for isotype, relative reactivity, specificity, antigen reactivity neutralization, and antibody pairing. |
| Delivery |
The typical delivery includes two vials of frozen cells or one flask of live cells and 5 ml of culture supernatant per clone. |
