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Antibody Generation and Validation

Antibody Generation and Validation

Conjugated antibodies for flow cytometry is an essential tool that requires validation

Andy Tay, PhD

Andy Tay, PhD is a freelance science writer based in Singapore. He can be reached at

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Antibodies, also known as immunoglobins, are produced by immune B cells in response to foreign molecules called antigens to neutralize them. They are used for numerous applications in biology and diagnostics.

Shaped like a Y, an antibody consists of the antigen-binding (Fab) variable region at the top, and a constant (Fc) region at the base. The Fab region enables an antibody to bind to a specific segment (or epitope) of an antigen through interactions like electrostatic forces and van der Waals forces. Attributed to their molecular recognition properties, antibodies are a popular tool in biological experiments. They are widely used in experiments to identify, isolate, and quantify specific protein antigens to understand their roles in physiology and diseases. Some examples include western blot analysis, immunostaining, and flow cytometry.

There are two main types of antibodies—polyclonal or monoclonal—and they are used in different settings. To detect the presence of multiple antigens for a simple yes/no diagnostic test, polyclonal antibodies have an advantage as they are able to bind to different epitopes. Hence, they are more tolerant of minor changes in antigen structure resulting from polymorphism and slight denaturation. On the other hand, monoclonal antibodies are more widely used in cases where specific antigen-antibody binding is crucial and cross-reactivity may invalidate experimental interpretation.

This article is a discussion of how antibodies are generated and the challenges associated with validation. The use of conjugated antibodies for fluorescence flow cytometry and cytometry by time-of- flight (CyTOF) applications is also explored.

Generating and purifying antibodies

Polyclonal antibody production exploits animal immune systems (often in rabbits or donkeys). Typically, the antigen of interest is repeatedly injected into the animals to evoke high expression of antigen-specific antibodies in the serum. For weakly immunogenic antigens, an adjuvant is also used to release the antigens in a sustained manner to increase the probability that an immune response will be elicited. Polyclonal antibodies can then be purified from blood serum using antigen affinity chromatography. The column matrix can either contain antigens against unwanted antibodies, allowing elution of the target antibody, or it can contain an antigen against the target antibody, allowing elution of unwanted antibodies.

Monoclonal antibodies can be produced using hybridomas. The antigen of interest is injected into a mouse to generate antibody-producing B cells. Using electro-fusion or chemicals like polyethylene glycol, the B cells will be fused with an immortal myeloma cell line to create hybridomas. Only myeloma cells that do not secrete antibody are used, and these cells also lack the hypoxanthine-guanine phosphoribosyltransferase (HGPRT) gene, making them sensitive to hypoxanthine-aminopterin-thymine (HAT) media that contains aminopterin that blocks DNA synthesis, and thus cell division. After electrical or chemical treatments, the cells are cultured in HAT media for 10 to 14 days and only hybridomas survive, as they contain the HGPRT gene from B cells while B cells with a shorter life span perish. The cell population is then diluted to one cell per well plate and as the antibodies in a well are produced by the same B cell cone, they recognize the same epitope, and are monoclonal.

Besides this method, advances in genetic sequencing have also given rise to recombinant antibody technology. Rather than using animals, the gene responsible for producing the target antibody is identified and cloned into an appropriate vector before being introduced into a host cell like Chinese hamster ovarian cell. The genetically-engineered host cell produces the monoclonal antibody, which can then be purified using methods like affinity chromatography.

Validating antibodies

Reduced antibody specificity, due to batch-to-batch variability, or antibodies that bind the wrong targets lead to poor experimental reproducibility, and can result in data misinterpretation and wasted resources. It is, therefore, important to check that the antibody is specific to its target antigen.

Related Article: Flow Cytometry: Fluidics and Versatility in Flow

There are a few simple ways to validate antibodies. First, labs should request characterization information of the same antibody batch being purchased from the manufacturer. Second, seek assistance and peer review from the community. If you are using an antibody that has been reported in a publication, it may be valuable to reach out to authors in that paper about their experience using the antibody. Initiatives like Antibodypedia and The Antibody Registry also list user-validated antibody information for references. Third, perform simple lab tests using positive and negative controls. Some examples include using cells with a knock-out gene expressing the antigen of interest or using siRNA to block the mRNA translation of target antigen.

Antibodies in flow cytometry

Fluorescence flow cytometry is a technique in which fluid-suspended fluorescently labeled single cells are flowed through a laser beam and the properties of the cells can be determined by their light scattering profiles. Fluorescence-tagged antibodies are used in flow cytometry to isolate and quantify cells expressing target antigens.

This technique relies on a primary antibody that targets specific antigens binding with a secondary antibody conjugated with fluorescence tags. The secondary antibody specifically binds to the heavy chain (Fc) region of the primary antibody. As an example, the primary antibody binds to antigen X and is produced in a rabbit. Then a suitable secondary antibody should bind to the Fc region of a rabbit immunoglobin and tagged with a fluorescence label (antirabbit-fluorescence). It should also come from a non-rabbit source and preferably an animal that shares little homology.

An emerging flow cytometry technique is cytometry by time of flight (CyTOF). This technique overcomes spectral overlaps in fluorescence flow cytometry, and it enables the detection of more antigens at a single time (approximately 100 antigens, compared to 40 antigens using traditional methods). In CyTOF, antibodies targeting antigens of interest are conjugated with metals instead of fluorescence tags. Antibody-bound cells are then sent through an argon plasma, which ionizes the metal-conjugated antibodies, and the metal signals are analyzed by a time-of-flight mass spectrometer. CyTOF is becoming a popular way to label intracellular proteins such as cytokines involved in complex signaling pathways that involve many proteins.

Antibodies are an indispensable tool in biology. Although the technology to manufacture them is established, antibody validation remains poorly standardized. Before using antibodies in experiments like flow cytometry, users should always perform a few extra steps to ensure they are validated.

For additional resources on antibodies, including useful articles and a list of manufacturers, visit