Time schedule for production of monoclonal antibodies and development of ELISA method

The time schedule is done in weeks as there could be slow delay if the immunisation is not good due to weak immunogenity of used antigen, or if hybridoma cells are growing very slowly. The difference in time could be on the end only 2 or 3 weeks.

 

 

Monoclonal antibodies

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

1

ELISA 1

Antibody captured assay.

We need only specific Ig produce by hybridoma cells.

2 3 weeks

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

Production of large scale Ig for protein A purification.

1

1000 mls of supernatant

Production of supernatant for large scale purification of Ig using protein A or protein L

3 weeks

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

Production of rabbit polyclonal sera for development of ELISA 2

1

Immunisation of rabbit

Production of polyclonal sera to supplied antigen of interest

10 weeks

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