The facility provides consultation to investigators in choosing the appropriate
peptides or proteins. Peptides should be conjugated to KLH for immunization and to BSA or OVA for screening by ELISA or Western Blot. In the case of the use of fusion proteins as antigen, small tags, e.g. hexa-histidine are recommended. Larger fusion tags like GST should be cleaved from the protein prior to immunization.
The facility will take responsibility for the ordering and the immunization of mice and rats. In general we begin with a 50 microgram (mice)/ 100 microgram (rats) priming dose of antigen followed by several boosting doses of 50/100 microgram, each . This regime will continue until a sufficient antiserum titre is achieved. Test bleeds are routinely taken and evaluated by the staff of the MAF using a direct binding ELISA. In addition, test bleeds will be sent to the investigators laboratory for evaluation in other assays. Data are evaluated jointly with the MAF staff to determine the performance of antisera at this point. Antiserum titres and specific assay performance must be acceptable to continue the project.
A fusion of lymphocytes from animals (1 or 2) which had most favourably responded to the immunization as determined by ELISA or alternative assay is performed. Lymphocytes are fused to Sp2/0-Ag14 cells using an optimized PEG-mediated fusion protocol. The fused cells are plated into wells of a stack of ten to twenty 96-well plates. Plates are monitored for growth and the supernatants of wells showing cell growth are screened by ELISA after 1 to 2 weeks. The screening procedure differentiates between IgG and IgM clones.
Cells from IgG-positive wells are transferred to 24 well plates, grown and screened again. The secondary screening verifies that the selected positive fusion products are still producing antibody. Supernatants from positive wells are then shipped to the investigator for evaluation in laboratory specific assays. The investigator then chooses the number of fusion wells for scale-up. It is important to account for the fact that not all wells that are scaled up will survive or produce specific antibody.
Cells from selected positive wells are then harvested and subcloned by limited dilution. Once colonies have been established, the respective supernatants are screened by direct ELISA assay. Subclones robustly secreting high titres of antibody are selected for freeze down and storage. The respective supernatants are shipped to the cooperation partners laboratory for further evaluation. All frozen subclones are stored in liquid N2 for several months/years.
Cells from growing antibody-secreting cultures can be expanded by progressive transfer to larger culture vessels to produce bulk antibody in a bioreactor or static culture.
Not only are the costs of the services provided by the MAF considerably lower than those of external commercial sources, but the MAF offers investigators the opportunity to be involved in every step of the antibody generation process. Once a hybridoma cell line is established, new batches of monoclonal antibodies of constant quality can produced by the MAF.
- If required the MAF also offers antibody purification using Protein G and Protein A chromatography.
- The MAF additionally offers cloning of the antigen encoding cDNA of interest into bacterial expression vectors followed by expression and purification of His-tagged recombinant protein for immunization purposes.