Usage example of monoclonal antibodies for extracellular vesicle markers

Yusuke Yoshioka – National Cancer Center Research Institute, Tokyo Japan – In 1975, Georges Jean Franz Köhler and César Milstein developed a technology of monoclonal antibody production and won Nobel Prize in Physiology or Medicine in 1984. This technology simply produces antibodies with single epitope but is still innovative effect to the field of medical or pharmaceutical industry for development of clinical diagnostic, antibody drug, molecular target drug and so on. Not only medical or pharmaceutical field, application of monoclonal antibody has been making a huge contribution to life science researchers. This is used for variety of applications such as detection of specific proteins, analysis of intracellular localization or protein-protein interaction and affinity purification thus it is important for researchers to obtain “good” antibodies which gives driving force to reach their goals. The word “good” contains variety of meaning but almost commons in sense; that is specific antibody recognizes target molecule specifically and bounds to single epitope in analysis.

In the field of Exosome research, importance of “good” antibodies is increasing. Exosome consists of membrane vesicles of 100nm and variety of proteins localize inside or surface of lipid bilayer. Currently, the methods of collecting Exosomes from biological fluid or cell culture supernatant and confirmation of their existence are immune electron microscopy or immunoblotting for Exosome marker proteins. It is essential to prepare “good” antibodies for these applications. In addition, ELISA method is applied to evaluate Exosome proteins and antibodies are also required for this purpose. Furthermore, antibodies are useful tools for immunoprecipitation or purification with affinity trap column for Exosome membrane proteins to collect specific Exosome populations.

Thus usage of antibodies for Exosome research ranges in many fields by applications but the most important thing is to select best antibodies recognize to specific target proteins. Exosome is a complex consist of variety of molecules and therefore does not always include a single protein. But it includes some specific proteins called “Exosome Markers” and these are common to most of Exosome vesicles. The existence of Exosome can be confirmed by detecting that of Exosome markers after collection of Exosome vesicles from sample although exact content of markers is not clear. In recent research, some kinds of Tetraspanin such as CD9, CD63 or CD81 are used for Exosome marker (1) and demand of antibodies recognize to these markers as antigen is increasing in proportion to its needs for research.

In researching Exosomes, it was extremely difficult to prepare “good” monoclonal antibodies recognizing to specific Exosome marker especially establishing their detection systems. Some clones used for trial could not show sufficient results. One of them was possible to apply to western blot but not suitable for detection of Exosomes, the main goal of the research.The difficulty in finding “good antibodies to specific Exosome markers had been long term stress for many researchers. But the problem has solved by appearance of new clone 12A12 to CD9 and 8A12 to CD63. These clones enable to establish detection system of Exosome showing sufficient result to the investigation. (Fig1)(2).These clones are also applicable to analysis with western blot (Fig2) or immune electron microscopy(3). 12A12 and 8A12 are very specific and recognize only for human antigens, not reactive to other spices such as mouse CD9 or mouseCD63. For this specific, these clones are not suitable to investigations with mouse but are effective to distinguish human exosomes from mouse subjects and will be valuable for cancer cell transplants of human to mouse. It is frequently heard about the rarity of “good” antibodies from researchers in many fields. Research of Exosome detection contains same problems. But things will be in progress in this field. Clones 12A12 (anti human CD9) and 8A12 (anti human CD63) will give bright light to the research of Exosome to be “excellent” antibodies for the detection.

1. Yoshioka Y, et al.: J Extracell Vesicles, 2: 20424, 2013
2. Yoshioka Y, et al.: Nat Commun, 5: 3591, 2014
3. Nishida-Aoki N, et al.: Mol Ther, 25: 181-191, 2017

Comparison data of Exosome antibodies

Experimental examole of western blot analysis for Exosome markers

Source – Cosmo Bio

Leave a Reply

Your email address will not be published. Required fields are marked *