Inject animal with antigens.

Upon contact with the antigen, the matching B cells mutate into centrocytes with varying affinity to the antigens. Through a repeated process of mutation and selection of higher affinity centrocytes, B cells with high affinity immunoglobulins are produced.

For more information, watch this video for: Antibody Affinity Maturation.

Isolate the cells from the animal’s spleen or lymph nodes. These lymphoid organs are rich in B cells.

Spleen B cells have the HGPRT gene, which is necessary for the hybridoma selection process.

Myeloma cells are immortalized cancerous B cells that have a mutated HGPRT gene and do not secrete their own antibodies.

Spleen cells (HGPRT⁺)(Antibody producing)

Myeloma cells (HGPRT^-)

Fuse spleen cells with myeloma cells in polyethylene glycol.

Unfused spleen cells

Fused spleen cells

Hybridoma cells

Fused myeloma cells

Unfused myeloma cells

Each hybridoma cell is immortal and expresses unique antibodies.

HAT medium (hypoxanthine, aminopterin, thymidine) selects against unfused cells.

Aminopterin inhibits the enzyme that is necessary for nucleic acid synthesis. The cells are forced to use alternative pathways to make nucleic acids by using HGPRT gene. Spleen cells are HGPRT⁺ and myeloma cells are HGPRT^- . Only when the spleen and myeloma cells are fused into hybridoma cells can they survive.

Select hybridoma cells in HAT medium.

Unfused spleen B cells will die out because they are mortal.

Unfused myeloma cells die because they lack the HGPRT gene.

Screen for positive, antibody-producing hybridoma cells by using the ELISA technique. For full details on this procedure, watch: Active ELISA Protocol

Antigen Reactive Hybridoma

Antigen Non-reactive Hybridoma

Coat with antigen.

Wash, then add hybridoma supernatant.

Add secondary antibody, then enzyme, and finally, substrate.

Upon addition of substrate, the enzyme produces a detectable color change in the solution.

Because this hybridoma's antibody never bound, the chain of reagents that followed also did not bind. Therefore, no color change can be observed.

Further screening is done depending on the final application the antibody is intended for.

After the screening process, the selected hybridoma cells go through additional steps:

Subclone and further screen the hybridoma cells.

Grow the hybridoma cells and purify the monoclonal antibodies.

Freeze and store the hybridoma cells.