Hybridoma technology utilizes a wide variety of experimental procedures to yield antigen-specific monoclonal antibody (mAb)-producing immortal hybridoma clones.

Conventional hybridoma technology utilizes in vivo immunizations, cell:cell fusion, specialized cell culture conditions, and various screening techniques to yield antigen-specific monoclonal antibody (mAb)-producing immortal hybridoma clones.  The concept is essentially unchanged from Kohler and Milstein’s original approach reported in 1975.  Animals (typically rodents) are immunized with the target antigen, splenocytes are fused with myeloma partners and grown in HAT media to select for hybridomas, and the hybridoma supernatants are screened for target reactivity.  Single cell cloning is performed to yield monoclonal antibody-producing hybridomas.

1. Rodents are typically immunized with antigen and adjuvant to generate a strong in vivo B cell antibody response.
2. The spleen is harvested and single cell suspensions of splenocytes are generated.
3. Splenocytes are fused with myeloma partners to generate hybridomas.
4. The fusion mixture is plated into 96-well plates where HAT culture media drives selection of hybridomas vs. unfused myelomas or B cells.
5. The plates are screened for antigen-specific antibodies.  Positive wells are expanded, and single cell hybridoma clones are generated from the parental hybridomas.
6. The clonal hybridoma is expanded for production of antigen-specific mAbs.