Hybridoma Selection

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.

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

hybridoma-infographic
figure-2-cell-fusion

Electrofusion efficiently fuses myeloma cells with splenocytes. Circle indicates fusion event. Myeloma cells: large nuclei; splenocytes: small nuclei.

figure-3-antigen-labeling

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.

plate-test-watermark

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.

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