DEVELOPMENT OF MONOCLONAL ANTIBODIES

Before beginning with the work, the specific characteristics of the antigen against which monoclonal antibodies are to be developed should be discussed with the customer. This will be helpful to verify whether i.e., specially designated screening methods for the specific mAk are necessary or already in place.

The antigens are provided by the customer. Peptides with less than 30 amino acids must possess an additional C- or N-terminal Cystein residue for coupling to a carrier protein. This must be considered in the course of the synthesis. For antigens with more than 30 amino acids, 2 mg are needed for the immunisations and 1 mg for the screening; for peptides with less than 30 amino acids, 5 mg peptide is needed for the coupling to a carrier protein und 1mg for the screening.

Custom monoclonal antibodies (mab) generation at UNICUS usually takes 5-6 months and is divided into 3 phases:

Work package 1: Immunisation (5-6 weeks)

  • Cross linking of the peptide to the carrier protein
  • Immunisation of three Balb/c mice incl. boosting and bleeding 
  • Detection of antibody titre in mouse serum by ELISA test to find the highest titre mouse to be used for cell fusion

Work package 2: Cell Fusion, Selection, Screening (app. 1 month)

  • Generation of the Hybridoma primary cultures by fusing the spleen cells of the selected mouse with the myeloma cell line SP2/0
  • Plating the fusion product into four 96-well plates for each fusion and cultivation in selective media
  • Screening of the primary cultures for wells producing specific IgG antibodies against the antigen applied by ELISA test
  • Transfer of the specific antibody-secreting hybridoma colonies to 24-well plates for cell propagation, retesting for specific antibodies
  • In case only work packages 1 and 2 are ordered by the customer, customer receives the repeated positive clones in 24-well or 48-well plates and a report on the work performed

Work package 3: Cloning, Re-cloning, Testing, Characterisation (app. 2 months)

  • Determination of the antibodies titre of the repeated high positive clones and selection of up to 8 primary cultures with a high level of antibodies titre for cloning
  • Cloning of up to 8 of the best cultures by limited dilution
  • Screening of the cloned cultures in ELISA, identification of the antibodies titre of the highest positive clones and selection of the cultures for the re-cloning
  • Re-cloning of up to 4 of the best cloned cultures, testing in the ELISA, determination of the antibodies titre and selection of the final clones
  • Cultivation and propagation of 2 final antibody positive stable re-cloned cultures
  • Determination of the Ig subclass and mycoplasma test
  • Cryoconservation
  • Customer’s delivery of cryo-stored clones on dry ice (3 vials for the two final clones, each with 3 x 106 hybrid cells) and upon request 10ml cell culture supernatant / cell line
  • Development of a detailed protocol and documentation

Following co-ordination measures to this effect, the customer can, in the meantime, receive   cell culture supernatant for each selected culture after primary screening, cloning and re-cloning for specific tests.

Prices on request.

All results of a project become the property of the customer after payment. The UNICUS Karlsburg OHG is obligated to refrain from either using the results of this development work itself or transmitting these results to any third parties. Strict confidentiality is considered part and parcel of the agreement.