Examples of monoclonal antibodies recognizing exosome markers

 

User Report

Yusuke Yoshioka

National Cancer Center Research Institute of Molecular and Cellular Therapy

Products

  • ●Anti CD9 for Exosome Isolation, Human (Mouse) Unlabeled, 12A12（Cat.No. SHI-EXO-M01）
  • ●Anti CD63 for Exosome Isolation, Human (Mouse) Unlabeled, 8A12（Cat.No. SHI-EXO-M02）
  • ●Anti CD81 for Exosome Isolation, Human (Mouse) Unlabeled, 12C4（Cat.No. SHI-EXO-M03）
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Experimental findings

In 1975 George Kohler and César Milstein invented monoclonal antibody production technology for which in 1984 they were awarded the Nobel Prize in Physiology or Medicine. Monoclonal antibodies have revolutionized the pharmaceutical industry, opening the door to new types of diagnostics and targeted therapies. It has benefited not only medical practice but also basic research. Not all monoclonal antibodies are equivalently useful; it is important to identify "good" antibodies for each specific application. In general, a “good” antibody is one that recognizes a single molecule and binds strongly to its molecular target.

Obtaining a "good antibody" is especially important in exosome research. Exosomes are lipid bilayer-delimited vesicles of approximately 100 nm in diameter that contain various proteins in their lumen as well as in their membranes. Antibodies have been used to detect and isolate exosomes occurring in body fluids or culture supernatants using immunoelectron microscopy, immunoblotting, ELISA, immunoprecipitation and immunoaffinity columns. These applications require “good” antibodies.

As exosomes are heterogeneous, it is likely that, depending on their source, they will display and contain distinct molecules. However, a subset of proteins is likely to be shared across different types of exosomes. Currently, the best pan-exosome markers are the tetraspanins CD9, CD63 and CD81. My exosome research began with the goal of building an exosome detection system. I struggled to find monoclonal antibodies that recognized exosomal markers in my detection system. Although I tried a number of clones, I did not obtain good results until I discovered anti CD9 (clone 12A12) and anti-CD63 (clone 8A12) (Figure 1 and Reference 2). These antibodies performed well in western blots (Figure 2) and immunoelectron microscopy. The human specificity of these antibodies was confirmed by their inability to detect mouse CD9 and mouse CD63. This property is useful in human xenotransplantation model systems in mice where the antibodies do not react with endogenous CD9 and CD63. If problems are encountered in detecting human exosomes, anti-CD9 (clone 12A12) and anti-CD63 (clone 8A12) antibodies may be useful.

Figures

Figure 1.ELISA performance of clones 12A12 (anti-CD9) and 8A12 (anti-CD63) relative to competitor clones A and B.
Figure 2.Western blot performance of clones 8A12 (anti-CD63) and 12A12 (anti-CD9) on human exosomes

References

  1. Yoshioka Y, et al.: J Extracell Vesicles, 2: 20424, 2013
  2. Yoshioka Y, et al.: Nat Commun, 5: 3591, 2014