High Affinity: Light Scattering Techniques for Antibody Characterization

The exquisite specificity and high affinity of antibodies for their antigen make them a precious therapeutic and diagnostic tool. Here we use dynamic, static and electrophoretic light scattering techniques to characterize a monoclonal antibody as well as antibody-coated gold nanoparticles.


An antibody is a complex protein produced by the immune system in response to a substance perceived as both alien and dangerous, termed antigen. When the antibody latches onto the antigen, it either directly neutralizes it, or “tags” it for elimination by other components of the immune system.

Antibodies are secreted by a class of white blood cells called B lymphocytes, or B cells. New B cells are constantly being produced in the bone marrow and, in their initial “naïve” state, all express a different antibody at their surface. During an infection, a naïve B cell whose surface antibody is capable of efficiently binding the pathogen will be triggered to rapidly multiply and maturate. The resulting clonal population of mature B cells then produces large quantities of soluble antibody. Mature B cells can later evolve into memory B cells, which can survive a lifelong in specialized niches in our immune system, and be reactivated by a later encounter with the pathogen.

The exquisite specificity and high affinity of antibodies for their cognate antigen has long been recognized and harnessed for biology and medicine. In biochemistry and molecular biology, antibodies are routinely used to detect and/or purify molecules of interest. In medicine, therapeutic antibodies are used as passive immunization against infectious agents, but also for the treatment of cancer (by targeting cancer-cell specific markers) and autoimmune diseases (by neutralizing the relevant inflammatory mediators). In diagnostics, the widely used lateral flow tests, used for instance as pregnancy tests or for the rapid detection of SARS-CoV-2 antigens, are based on the recognition of the target by a specific antibody coupled with a colored reagent, most commonly gold nanoparticles.

Creating and manufacturing antibodies is a highly complex process, and the resulting product, like most multimeric proteins, is a very fragile molecule. Specificity, purity and conformational integrity all need to be assessed during the quality control process. Here we demonstrate how the Litesizer 500 instrument can help determine the purity and integrity of monoclonal antibodies by assessing their particle size and molecular mass. We also show how the Litesizer 500 can be used to optimize the labelling of antibodies with colloidal gold, e.g. for the development of lateral flow tests, through the determination of the antibody’s isoelectric point and the sizing of the conjugates.

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