Hybridoma Antibody Engineering


Hybridoma cell lines offer a time-tested way to engineer custom monoclonal antibodies (mAbs). Developed in 19751, hybridoma cell lines are the result of a fusion between a B cell and an immortal Myeloma cell. Though the hybridoma cell line creation workflow is a proven procedure, it takes time and many hands-on inputs. Hybridoma creation requires isolating B cells from an immunized animal, fusing a B cell with a myeloma cell, and evaluating your new hybridoma cell lines for antibody quality and production efficiency. Hybridoma cell lines present their own challenges for antibody engineering, being particularly labor-intensive and requiring a long time to develop. Do not write them off as a technology of yesteryear. Many perceived disadvantages of the hybridoma workflow can be alleviated through instrument automation and integration.

hybridoma antibody engineering workflow overview with product call-outs 

B Cell Isolation

Challenge: Isolate a substantial population of B cells while preserving cell viability

Starting with a large pool of B cells is ideal for hybridoma creation. Donor animals, typically mice, are injected with an antigen to create an immune response. Immunized animals are bled to test for appropriate antibodies. If the test bleed is positive for an immune response, spleens are harvested and B cells extracted by density centrifugation or flow cytometry. B cells are mortal and do not tolerate culture. They must be directly used or cryopreserved.

Cell fusion and selection

Challenge: Create and culture hybridoma cell lines efficiently

B cells and immortal Myeloma cells must be fused to create a hybridoma cell line. B cells and Myeloma cells are co-cultured in a medium containing polyethylene glycol or electrofused, inducing cellular fusion and creating a hybridoma cell line. After fusion, cells are cultured in hypoxanthine-aminopterin-thymidine (HAT) medium. Only successfully fused hybridomas survive.

Hit picking and sub-cloning

Challenge: Refine cell line possibilities and verify antibody production

After fusion, hybridomas must be tested to ensure they are producing an antigen-specific monoclonal antibody. A single hybridoma cell is cultured and allowed to produce antibodies to screen with ELISAs or other methods. As these single lines grow, they are serially diluted selecting for the fastest-growing and most productive cell lines. Parallel culture of so many new hybridoma cell lines requires hands-on input and strict data management. We provide solutions for automated cell culture and cell line maintenance while integrating data capture2.


  • Hybridoma cell line creation offers a cost-effective way to produce mAbs that has stood the test of time.
  • B cells must be isolated from immunized animal spleens and do not tolerate culture. We can expedite the extraction procedure with automation so B cells are ready to use quickly2.
  • Immortal Myeloma cells and B cells are fused to create a hybridoma cell line. Automate cell fusion and post-fusion selection to reduce hands-on inputs.
  • Hybridoma cell lines are cultured and monitored for quality and robust mAb expression. Many cell lines are cultured in tandem. We offer an integrated culture and data collection solution that can be tailored to your needs2.


  1. Kohler, G. & Milstein, C. 1975. Continuous cultures of fused cells secreting antibody of predefined specificity. Nature. 256, 495-497.
  2. AAG-345101.18


Products and methods described are not intended for use in diagnostic procedures.