Custom Immunology Services
Rat Hybridoma Development |
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Covance has generated hybridomas against a large variety of antigens. Prior to project initiation, you are provided with personalized project proposals and strategy consultation. Hybridoma cell lines developed by Covance remain exclusive property of the client. A typical hybridoma development project usually takes 6-8 months and has four phases: immunization, fusion, cloning and hybridoma stabilization. Any aspect of these four phases can be customized to meet your needs. The investigator has the option to terminate hybridoma development projects prior to the beginning of any phase. If desired, projects can be transferred to you at any point during these phases to give unparalleled control over your project. Our standard schedule of event for a 5 rat project requires 2,950 µg of immunization antigen or 590 µg / rat. Optimally, we need about 500 µg of antigen for screening by ELISA.
Phase I: Immunization
As a general procedure we immunize 5 female Sprague Dawley rats with immunogen emulsified in Freund's adjuvant. Subsequent injections follow a three-week cycle in which blood samples are drawn ten days after every injection. Serum samples are titered by ELISA. Client selected rats are hyper-immunized with low dosage boosts and are then used for the fusion.
Immunogen Requirements:
- Immunogen may be sent in liquid, powder, or gel format. The immunogen will need to be soluble (no gel) for the hyper-immunization at the end of the schedule.
- The recommended concentration is 1.0 mg/mL or greater and the recommended buffer is PBS.
- An additional 500 µg of antigen suitable for plate coating is required for ELISA testing, i.e. soluble in PBS.
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| Rat Immunization Phase |
| Day 0 |
Sprague Dawley Rat — Female
Pre-bleed (avg. 0.5 mL serum)
1° IP: 200 µg with FCA |
| 21 |
Boost IP: 100 µg with FIA |
| 42 |
Boost IP: 100 µg with FIA |
| 52 |
Test Bleed (avg. 0.5 mL serum) |
| 53 |
ELISA (5 rats) |
| 63 |
Boost IP: 100 µg with FIA |
| 73 |
Test Bleed (avg. 0.5 mL serum) |
| 74 |
ELISA (5 rats) |
| 84 |
Boost IP: 100 µg with FIA |
| 94 |
Test Bleed (avg. 0.5 mL serum) |
| 95 |
ELISA (5 rats) |
| 98 |
Boost IV: 40 µg (pre-fusion rat) |
| 101-110 |
Phase Conclusion* (avg. 7.0 mL serum, pre-fusion mouse) |
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| *Contact us for more details regarding phase conclusion. |
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Phase II: Fusion
After immunization, spleens of the hyper-immunized rats are prepared and the spleen cells fused with rat derived YB2/0 myelomas using polyethylene glycol. Viable hybridomas are selected by treatment with HAT media and screened for secretion of antibody to the antigen by ELISA. The antibody secreting hybridomas (up to 48) with the highest absorbance determined by OD measurement are selected for expansion and cryopreservation into duplicate vials. Media (~500 µL) is shipped to the client for evaluation in the client's specific assay. |
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| Rat Fusion Phase |
| Day 0 |
Fusion of spleen cells with myelomas |
| 14 |
Clones selected and incubated in controlled environment for 1-7 days |
| 18-21 |
ELISA (Ten 96 well plates) |
| 21-35 |
Expansion and freezing of up to 48 hybridomas |
| 36-42 |
Shipment of media |
Note: Any remaining spleen cells may also be frozen.
- Client may also chose to freeze spleen cells from any immunized rats as a back up for future hybridoma development.
- Additional antibody containing culture and ELISA screenings can be requested for additional charges.
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Phase III: Cloning
A maximum of 5 positive primary clones selected by client will be cloned by serial dilution and plated on two 96 well plates each. Growing wells will be screened for secretion of antigen specific antibody by ELISA. The media samples (~500 µL) from up to 10 positive clones with the highest absorbance determined by OD measurement are shipped to the client for evaluation and the clones are temporarily frozen into duplicate vials. |
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| Rat Cloning Phase |
| Day 0 |
Cloning procedure |
| 14-16 |
ELISA (Ten 96 well plates) |
| 16-30 |
Expansion and freezing of up to 25 hybridomas |
| 31-42 |
Shipment of media |
| Note: Additional cloning as well as isotyping of selected clones is available. |
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Optional but Recommended:
Phase IV Stabilization of Hybridoma
A clone of your choice from Phase III will be cloned again by limiting dilution to generate a stable, third generation cell line. The growing cells are screened for antigen specific antibody by ELISA. The media samples (~500 µL) from up to 10 positive clones with the highest absorbance determined by OD measurement are shipped to the client for evaluation and the clones are temporarily frozen into duplicate vials. One cell line is selected for final expansion for long term storage or scale up by in vitro methods or ascites production. |
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| Rat Stabilization Phase |
| Day 0 |
Cloning procedure |
| 14-16 |
ELISA (Five 96 well plates) |
| 16-30 |
Expansion and freezing of up to 10 hybridomas |
| 31-42 |
Shipment of media |
| Note: The option to re-thaw the re-clones for expansion and cryopreservation of 10 vials each is available. |
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Although we realize that many companies are not offering this final stabilization phase, our experience reinforces its importance. We do recommend it to provide you with the most stable, reproducible cell line for continued, reliable use in your research.
Download needed order forms. We'll process your order immediately.
Our Shipping Address
Attn: Immunology Services
465 Swampbridge Road
Denver, PA 17517
tel (800) 345-4114
tel (717) 336-4921
fax (717) 336-3481
AAALAC International Accredited, GLP Studies, OLAW Assured, USDA Research Registered
Document Revision Date: 6/12/07 |
