Detection of exosomes in various biological fluids utilizing specific epitopes and directed multiple antigenic peptide antibodies

Abstract

Exosomes are nanosized cell-derived vesicles that have recently gained attention for their use as a biomarker since the biomolecules encountered in these vesicles are directly linked to specific diseases including immuno-inflammatory, cardiovascular, and hepatic disorders. Furthermore, due to their nano size and safe travel in extracellular fluids, exosomes have been investigated as natural drug delivery systems, delivering cargo to destined cells with excellent specificity and efficiency, and crossing the blood–brain barrier. This necessitates the isolation and detection of exosomes. However, numerous exosome isolation techniques are available, including ultracentrifugation, size-based chromatography, polymer precipitation, microfluidics, and immunoaffinity-based isolation, with the downfalls of non-specificity and lower cost-effectiveness. This article introduces an immunoaffinity-based detection of exosomes using targeted anti-exosome antibodies raised in chickens due to its economic and commercial viability. The current study is unique in that it identified a specific antigenic region on exosomal surface tetraspanins (CD9, CD63, CD81) and constructed a multiple antigenic peptide dendrimer for making a small peptide as an immunogen without the use of a carrier protein. The antigenic region selection is critical to the study because it determines the efficiency of antibodies for exosome capture. This technique was validated using enzyme-linked immunosorbent assay in various biological fluids such as serum, urine, milk, plasma, and blood due to its numerous advantages including high sensitivity, specificity, handling multiple samples at once, requiring a small sample amount, and no purification as an antigen. In light of this technique, it is a useful tool for clinical monitoring of the patient’s biological conditions.

Efficient strategy to isolate exosomes using anti-CD63 antibodies conjugated to gold nanoparticles

“…Based on the evaluation of earlier studies for protein purification, the isolation and purification of antibodies from the immunized chicken were carried out using the egg yolk component (Sunita et al 2020 ; Panwar et al 2023 ). In a nutshell, egg yolk from immunized chicken eggs was diluted in deionized water at a 1:10 ratio and homogenized to make a uniform suspension.…”

Exosome-Like Systems: From Therapies to Vaccination for Cancer Treatment and Prevention—Exploring the State of the Art

“…Tetraspanins family (CD9, CD81, CD63, CD82) [40,41] Immunomodulatory (MHC-I, MHC-II, PDL-1) [42][43][44][45] Lipid raft (PS, Sphingolipids, Cholesterol, Ceramide) [46] Luminal Space…”

The Role of Bacterial Extracellular Vesicles in the Immune Response to Pathogens, and Therapeutic Opportunities

“…Classic exosomes express the tetraspanins CD9, CD63, and CD81, while non-classic exosomes express CD9 and CD63, but not CD81 [ 16 , 20 , 21 , 22 ]. Classic microvesicles express ADP-ribosylation factor 6 and CD35 and bind annexin A1 [ 16 , 21 ], while apopototic EVs bind annexin V [ 23 , 24 ].…”