BioLegend Making Antibodies
Antibodies are immunoglobulins, abbreviated as Ig, made by the body's adaptive immune system. Through complex maturation processes, specific antibodies are created to target antigens. This webpage discusses how antibodies are naturally made in the body and how they can be generated for research use.
Creating Monoclonal Antibodies
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.
Creating Polyclonal Antibodies
1. Multiple injections with antigen help to boost the antibody titer.
2. Polyclonal antibodies are affinity purified from the animal serum.
3. The polyclonal antibodies are then tested in various applications.
Antibodies In Vivo
B cells mature in the bone marrow and create antigen-specific antibodies.
B cell maturation involves class switching and creation of antigen specificity. Antigen specificity is created by the random rearrangement of genes that code the antibody.
When the B cells first encounter the matching antigen, the B cells become highly specific from antibody affinity mutations.
B cells differentiate into either memory B cells or plasma cells.
Memory B cells live longer and express the same antibody as the parent.
Plasma cells produce mass amounts of altered antibodies that recognize the same antigen.
For more information, see our Maturation Markers page.
Antibody Structure
An antibody is roughly shaped as a ”Y“, consisting of 2 heavy chains and 2 light chains. Mouse over the parts of the antibody to learn more.

Functions of Antibodies:
  • Neutralizes toxins by binding to them
  • Directs phagocytosis and killing of foreign microbes
  • Mediates complement killing of foreign cells, bacteria, or viruses
For more information, view this helpful Youtube video: What are Antibodies?
binding lightChain heavyChain
Two types of light chains: kappa (κ) and lambda (λ)
Five types of heavy chains:
  • IgG – most abundant Ig and activates classical complement pathways
  • IgM - large agglutinating pentamer that is the first response to antigens
  • IgD - expressed by mature B cells and bound to B cell membranes
  • IgA - found in external secretions such as tears, mucus, and saliva
  • IgE - involved in allergic reactions
The antigen binding site is formed by both the light and heavy chains and is complementary to the epitope of an antigen.
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