Monoclonal antibody projects are split into three initial phases with several additional stages also available to the customer if necessary:

Stage 1 - Immunisation of 4 hosts
Stage 2 - Fusion & initial screening and selection of positive hybridomas
Stage 3 - Cloning to produce up to 6 viable cell lines

Stage 1 - Immunisation of 4 hosts

Four Balb-c hosts will be immunised with the customer’s antigen. At six weeks, following the primary immunisation and the first boost, the hosts will be tested for antibody titre. The antibody response will be determined by ELISA, unless the customer has an alternative assay. If additional boosts are required, they will be administered and the hosts will be retested for an antibody response. Once immunisation is complete, the hosts will be held for three to four weeks before performing the hybridoma fusion. Three days before the fusion, a single host will be selected for the pre-fusion boost.

Stage 2 - Fusion & initial screening and selection of positive hybridomas

A spleenectomy is then performed and the spleen cells fused with a myeloma cell line. We use up to 10 96-well panels for plating. Fused cells are grown in HAT selection medium and all 96-well panels are screened by ELISA for antibody positive wells. The ELISA positive wells (maximum 100) are then expanded and retested three to four days later by ELISA. The standard production is screened with a single positive and negative antigen. Additional ELISA antigens may be added as an option. A maximum of 30 positive wells are frozen as a back up and 0.5ml of tissue culture supernatant/clone can be sent to the customer for further testing at this point.

Stage 3- Cloning to produce up to 6 viable cell lines

First cloning – A maximum of 6 positive wells tested will be selected for cloning by limiting dilution. Isolated clones will be tested by ELISA and positives are selected to expand for the next cycle of cloning. Cells from each of the clones are frozen in triplicate as back up.

Additional Services

Final Cloning

Approximately six positive clones from the first cloning cycle will be selected for the second cycle. Several cycles of cloning may be required in order to obtain a true, stable clone. Isolated colonies will be tested by ELISA and positive selected to expand. Cells from these positive clones will be frozen in triplicate as back up.