Phase |
Procedure |
Description of used procedure |
Time of realisation |
1 |
Immunisation |
5 mice immunised using 50micrograme of antigen (4injection per mice) |
6 - 8 weeks |
2 |
Cell-fusion |
Fusion of splenocytes and myeloma cells |
Week 7 or week 9 |
3 |
Screening |
Screening of hybridomas for production of non-specific Ig and recognition of specific antigen |
Week 10 |
4 |
Propagation of hybridomas |
Cryopreservation and production of cell bank |
Week 11 |
5 |
Recloning |
Recloning of all positives hybridoma cells and Ig (IgG1, IgG2a etc.) characterisation |
Week 11 and week 12 |
6 |
Expansion |
Production of 50mls of supernatant from each clone and preparation of cell bank for each cloned hybridoma |
Week 12 and week 13 |
Development of ELISA |
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1 |
ELISA 1 |
Antibody captured assay. We need only specific Ig produce by hybridoma cells. |
2 3 weeks |
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2 |
ELISA 2 |
Two antibody sandwich immunoassay. We need to develop the polyclonal sera to the studied protein |
4 6 weeks if rabbit sera is available |
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Production of large scale Ig for protein A purification. |
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1 |
1000 mls of supernatant |
Production of supernatant for large scale purification of Ig using protein A or protein L |
3 weeks |
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2 |
Protein A or L purification |
1-2 mgs of purified specific Ig from one supernatant including Ig concentration measurement and purity determination using SDS ELFO |
1 week |
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Production of rabbit polyclonal sera for development of ELISA 2 |
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1 |
Immunisation of rabbit |
Production of polyclonal sera to supplied antigen of interest |
10 weeks |
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2 |
Protein A purification |
Purification of Ig from rabbit sera using protein A column including Ig concentration measurement and purity determination using SDS ELFO |
1 week |
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