Generation of Monoclonal Antibody and Production

Service protocol

 

Phase I (immunization phase)

Includes immunization of up to three mice (Balb/C) with supplied and purified antigen, collection of preimmune and immune sera and ELISA based analysis. The customer will decide which immune-responsive mice will be used for subsequent fusion.

Phase II (fusion phase)

This procedure includes the production of primary cultures, selection on HAT and HT medium and screening. All primary cultures will be tested for positive clones secreting IgG or other isotypes with specificity of the respective immunogen. Up to two fusions per antigen are included.

Phase III (subcloning phase)

Up to ten positive primary clones will be cloned by a maximum of two limiting dilution cloning steps and screened by standard ELISA. This procedure takes part in an automated robotic system, including an integrated imaging unit to monitor clone performance. For example, the monoclonality of the selected clones can be verified via backtracking of the images. This will reduce time and costs for the generation of monoclonal cells. The customer will receive 1 ml supernatant of each positive clone to confirm the results. From each selected positive clone three aliquots will be stored in liquid nitrogen at customer’s request. One aliquot of each clone remains as a backup in the contractor’s laboratory, which will not be used for other purposes or distributed. Upon request, additional cryo-cultures can be delivered. All selected clones from Phase III will be further characterized by determination of IgG class/subclass and mycoplasma test.

Phase IV (expansion/purification)

Upon request, we can expand up to two selected positive clones and purify mAbs from the supernatant of 1 liter culture supernatant. Furthermore, we can provide mAb production in perfusion or fedbatch fermentations. At the conclusion of a successful project, all remaining mice will be sacrificed. If the customer accepts the mouse housing charges of € 50 per month, mice will be kept for possible additional fusions or isolation of mRNA from spleen cells.

The success of the hybridoma fusion and delivery of antigen specific mAbs is dependent on the immunogenicity of the antigen supplied by the customer. We cannot guarantee that the antigen supplied by the customer will produce an immune response sufficient to warrant of the generation of hybridoma cell lines and mAb production/purification.

Please request for prices and conditions.