Step by Step Guide to Creating Custom Monoclonal

Generation of producing Is the mouse producing your 1 B Cells in mice 2 antibodies of interest?

Antibodies

Epitopes

Blood Antibodies Sample of Interest

Antigens Host Animal Host Animal (Mouse) (Mouse)

A mouse is immunized against the target antigen of interest Test bleeds are collected and analyzed for the presence of using, antigenic peptides, proteins or biological material NO antibodies targeting the antigen of interest. that is prepared with a suitable adjuvant and used under a specified immunization protocol. YES

Dissociation of mouse tissue 3 releasing resident B cells and mixed 4 Addition of Polyethylene Glycol (PEG) with a specific ratio of immortal mouse myeloma cells Myeloma Cell Myeloma Cells

Hybridomas

Spleen

B Cells BCells Myeloma Cells Fusing issociate Splenocytes

Mouse splenocytes are mixed with a specific mouse After PEG is added to the cell mixture, certain cells fuse myeloma cell line. to form hybridomas.

Selection of fused hybridoma cells Selection of fused hybridoma cells and 5 from unfused B Cells 6 unfused myeloma cells

Hybridomas Hybridomas Myeloma Cell

Unfused Unfused B Cell Myeloma Cell ying ying

Unfused B Cells will not survive in culture, leaving only the HAT Medium allows fused hybridomas to thrive, while hybridomas and the unfused myeloma cells. unfused myeloma cells lack an enzyme that allows them to survive in HAT Medium

7 Clonal selection and expansion 8 Hybridoma screening

Plate Bound Antigen of Interest in Each Well

After weeks of cell culture expansion, the conditioned media is Hybridomas that survive are separated into single cell screened for the presence of secreted antibodies specific for cultures, one cell per well. the antigen of interest by Flow Cytometry or ELISA.

Positive hybridoma selection and Antigen specific monoclonal 9 expansion 10 antibodies are scaled up and stored for additional testing

Positive Test Culture Flask

Plate Bound Antigen of Interest in Each Well

Positive Screening by Flow Cytometry or ELISA result in Antigen Finally, Monoclonal antibodies are grown in large scale specific Monoclonal Antibodies. and then frozen for long term storage.

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