Antibodies (Exosome)

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Marker Proteins of Extracellular Vesicles (Including Exosomes)

Extracellular vesicles (EVs), such as exosomes, are known to contain marker proteins, including the tetraspanin family (CD9, CD63, CD81), integrin family (ITGA, ITGB), ESCRT complex-binding proteins (TSG101, ALIX), lipid raft markers (Flotillin-1/2), and heat shock proteins (HSP70). The expression levels of these marker proteins vary depending on the cell of origin; in some cases, certain marker proteins may be minimally expressed.

In this context, the International Society for Extracellular Vesicles (ISEV) published a position paper, MISEV2018, in 2018¹⁾, proposing proteins useful for demonstrating the presence and purity of EVs (Table 1). The paper highlights the importance of confirming not only the presence of EV proteins (Categories 1a or 1b, 2a, and optionally 2b in Table 1*) but also, at a minimum, any impurities such as lipoproteins and albumin (Categories 3a or 3b in Table 1). Category 4 pertains to proteins specific to certain small EVs, and Category 5 includes proteins required for analyzing the functions of EVs.

Table 1: Examples of Marker Proteins Used for Characterizing EVs by Protein Constituents (Partial excerpt from MISEV2018)

Production of Antibodies Against EV Markers

Among the marker proteins of EVs, membrane proteins such as CD9, CD63, and CD81 are challenging targets for producing high-quality antibodies using conventional methods due to their complex tertiary structures. To address this issue, Fujifilm Wako produces anti-CD9, CD63, and CD81 antibodies using the “DNA immunization” method.

Figure 1. DNA immunization

DNA immunization involves the administration of an expression vector containing the cDNA of the target antigen for animal immunization, rather than using peptide or protein antigens. The administered cDNA of the target antigen is transcribed under the control of a promoter in the vector and expressed within the animal's body, producing an antigen. Antibodies produced by this method primarily recognize antigens in their native form, enabling the production of antibodies with high affinity even against membrane proteins with complex tertiary structures (Figure 1).

In practice, using Fujifilm Wako’s “Anti CD9, Monoclonal Antibody (1K)” (Product Number: 014-27763), CD9 in EVs purified from COLO201 cell culture supernatant was detected by Western Blotting under non-reducing conditions. This antibody demonstrated a higher sensitivity in detecting CD9 compared to conventional products (Figure 2). Similar results were obtained with Fujifilm Wako’s anti-CD63 and anti-CD81 antibodies, further indicating their value as research tools for EVs, including exosomes.

Figure 2. Performance Comparison of Anti-CD9 Antibodies by Western Blotting

EVs were purified from COLO201 cell culture supernatant using the PS affinity method, followed by analysis of CD9 by Western blotting.
Primary Antibody: Anti CD9, Monoclonal Antibody (1K)
Secondary Antibody: Anti Mouse IgG (H+L), Peroxidase-conjugated
Detection Reagent: ImmunoStar^® Zeta (Product Number: 291-72401)

References

1. Théry, C. et al.: J. Extracell. Vesicles, 7(1), 1535750(2018).
   Minimal information for studies of extracellular vesicles 2018 (MISEV2018): a position statement of the International Society for Extracellular Vesicles and update of the MISEV2014 guidelines