Our Smart Selection Approach
Hybridomas are immortal somatic cell hybrids that secrete antibodies. Hybridoma generation is a critical step in making monoclonal antibodies. A major barrier to monoclonal antibody discovery is hybridoma selection. Hundreds to thousands of hybridoma cells have to be manually screened in the hope of identifying antibody-secreting clones.
BiCell Scientific Inc has developed a “smart” selection approach to identify antibody-secreting hybridoma cells and streamlined the process for monoclonal antibody production. Our "smart" selection approach is included into monoclonal antibody production service in Custom Antibodies.
Flow chart of “smart” hybridoma selection
Step 1:
Mouse is immunized with antigen
Step 2:
Splenocytes from immunized mouse are isolated
Step 3:
Splenocytes are fused with myeloma cells (our electrofusion protocol can generate 1 hybridoma clone out of 104 splenocytes)
Step 4:
Hybridoma cells are treated by a proprietary crosslinking protocol to immobilize antibodies on the cell surface
Step 5:
Fluorophore labeled antigen molecules allow identifying positive hybridoma cells by direct binding to the hybridoma cell surface
Step 6:
Fluorescence based cell sorting
Step 7:
Dilution cloning and clonal selection
Electrofusion efficiently fuses myeloma cells with splenocytes. Circle indicates fusion event. Myeloma cells: large nuclei; splenocytes: small nuclei.
Antigen labeling of hybridoma cells. Hybridoma cells are transiently crosslinked to immobilize antibodies on the cell surface. FITC-labeled antigen binds to and decorates the hybridoma cell that secretes the correct antibody.
Hybridoma cell plating and screening. Antibody secreting hybridoma cells are cultured and their supernatants screened in 96-well plates. Over 50% of hybridoma cell clones show positive binding to antigen. Procedure: plate was coated with peptide antigen at 1ug/ml in PBS, followed by incubation with 50ul supernatant for 1hr and secondary antibody (HRP) for 1hr. HRP substrate (blue color) develops within 5 min. No acid was added. Image was taken with iPhone.
Our Workflow Chart
| Steps | Timeline (weeks) | Deliverables/Internal testing milestone | QC standard |
| Phase I: Antigen preparation (peptide antigen,13-19 aa length, will be designed against target protein and its region of interest, synthesized and conjugated to KLH) | 2 weeks | / | COA
|
| Phase II: Animal immunization (3 Sprague Dawley rats or 5 Swiss Webster mice) will be immunized with antigen; conventional protocol – three injections)
| 8-10 weeks | 100 ul anti-serum | ELISA after 3rd injection |
| Phase III: Cell fusion and screening (fusion of mouse myeloma cells with splenocytes from one best responder animal to generate hybridoma and perform antigen based hybridoma selection, enriching, and plating)
| 4-6 weeks | Supernatant from 96 wells | ELISA from supernatant |
| Phase IV: Expansion and antibody purification (customer choosing one clone; expanding clone and purifying antibodies with affinity chromatography)
| 6-8 weeks | One hybridoma cell line (to customer); purified monoclonal antibody from the selected hybridoma cell line (1mg) (BiCell Scientific will bank all remaining clones; customer can request additional clones by paying a service fee for hybridoma recovery) | ELISA from supernatant |