Phase 1: Immunization
Immunization of Balb/c mice ( up to 5 animals) with antigen to stimulate antibody production. You may choose to follow our standard immunization protocol, or work with our staff to use your own protocol. We typically inject mouse every two weeks for 6 weeks before taking a test bleed. The test bleed serum samples are analyzed for antibody production using ELISA. If desired, clients can request up to 0.5 ml serum sample/per mouse for their own additional characterization. ELISA will be used to determine the highest titer mice to be used for cell fusion. Immunization may continue longer if response is not optimal.
· 5 mg of purified recombinant protein or intact protein will be needed for immunization.
With peptide synthesis request: Custom Peptide synthesis up to 15 amino acids (additional amino acid for $15 per residue), HPLC purification up to 90% purity, peptide KLH conjugation for immunization, peptide BSA conjugation for screen and 10 mg free peptide will be provided for client.
Lead time: 8-10 weeks
Phase 2: Fusion
Fusion of spleen cells with myeloma cell line. The fused cells are then placed in several 96-well culture plates. Viable colonies appear as early as 3-5 days after fusion and are kept healthy with a fresh supply of nutrient media as necessary
Lead time: 2-6 weeks
Phase 3: Screening
When hybridoma colonies are large enough to screen, screening of all wells containing fusion products is done with ELISA assay. All positive wells will be expanded and frozen for parental hybridomas. The number of positives depends on the nature of the antigen, and can range from a few hybridomas to a dozen or more. Additional hybrids can be frozen for further studies.
Lead time: 2-6 weeks
Phase 4: Stabilization and Expansion of clones
After positive hybridomas are selected for further development, they will be cloned using the limited dilution method and screened by ELISA. 5 clones with the desired specificity will be expanded, and hybridoma medium (50 – 100 ml) will be collected for further studies
Lead time: 2-4 weeks
Phase 5: Isotyping of clones and Ascites Production
Isotypes of all selected clones will be determined and production of ascites from 5 mice if needed
Lead time: 3-5 weeks
Total time for project: 17-29 weeks
The secreted antibody will be purified by Protein G affinity chromatography and send to the customer. Approximate yield may be 20-30 mg. Antibodies are eluted using both pH11 and pH2.5 buffers and neutralized immediately followed by dialysis in PBS.
Lead time: 1 week