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

Dikshita Panwar; Deepali Shrivastava; Shalaka Bhawal; Lavleen Kumar Gupta; N. S. Sampath Kumar; Anjani Devi Chintagunta

doi:10.1515/revac-2023-0056

Article Open Access

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

Dikshita Panwar , Deepali Shrivastava , Shalaka Bhawal , Lavleen Kumar Gupta , N. S. Sampath Kumar and Anjani Devi Chintagunta

Published/Copyright: May 12, 2023

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From the journal Reviews in Analytical Chemistry Volume 42 Issue 1

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.

Graphical abstract

Keywords: exosomes; ELISA; immunoaffinity; multiple antigenic peptides; tetraspanins

1 Introduction

Extracellular vesicles (EVs) are cell-secreted vesicles including exosomes, macrovesicles, and apoptotic bodies. EVs were first noted as being involved in cell–cell communication in the 1990s. More recently, they were found to modulate the immune system, maintain tissue homeostasis, and control disease pathophysiology [1]. Exosomes are secreted by a variety of cells, including cancer cells, immune cells, endothelial progenitor cells, and mesenchymal cells, and are capable of transferring biomolecules that change the fate of the recipient cell [2]. Exosome research has advanced significantly over the past few years as a result of their nano size (30–150 nm) and numerous important roles in cardiovascular disease, tumour development, immune response regulation, nervous system development, and most importantly, mediating the inflammatory response [2]. Furthermore, in order to overcome the limitations of tissue biopsy, which is less assessable for deep tumour cells and tricky to characterize multiple tumours, these are used in liquid biopsy and are regarded as circulating biomarkers in therapeutics [2]. Exosomes are secreted by most cell types in physiological and pathological conditions, including dendritic cells, lymphocytes, mast cells, and epithelial cells, and are released into various biological fluids such as blood, urine, saliva, and milk [2,3]. These are loaded with a parent cellular cargo that alters in diseases like cancer, rheumatoid arthritis, obesity, kidney disease, and so on, and the receiver cell stimulates by transferring activated receptors and surface-bound ligands [2,4]. Exosomal biogenesis differs from other EVs as it begins with plasma membrane internalization and progresses to the endosome, where it forms intraluminal vesicles via inward budding and is collected in the late endosome, where it is referred to as multivesicular bodies (MVBs) [4]. Through budding, MVBs are released from the late endosome and fuse with the cell membrane for secretion as an exosome into the extracellular environment [4]. Exosomes have late endosomal receptors including CD9, CD63, CD81, heat shock proteins (HSP70), Alix, and so on, due to their endosomal origin [3,4]. These receptors have been used for immunoaffinity extraction of exosomes from various biological fluids and serve as potential exosomal surface makers. Being such a valuable asset in research, it is unfortunate that there is no standard isolation of exosomes other than the gold standard ultracentrifuge, which has the downside of compromising exosome purity with other molecules of similar size [4]. Other particle size-based exosomal isolation methods include ultrafiltration, size-exclusion chromatography, and precipitation, which results in a mixed population separation. Due to the presence of various proteins and receptors on the surface of exosomes, it is possible to develop a highly specific immunoaffinity technique for exosome trapping [5]. Another isolation method, microfluidics, is being established as a specific tool for exosome isolation, despite its numerous limitations including high device complexity, chip limitations, and high production cost [5,6]. In this article, we have worked on developing a technique to detect exosomes with an easily available, well-established, and low production cost method, which is enzyme-linked immunosorbent assay (ELISA). For this purpose, anti-exosomal antibodies raised against the exosome surface tetraspanins CD9, CD63, and CD81 were used to develop the immunoaffinity technique.

To elicit antibodies, a molecule must meet two basic requirements: it must be foreign in order to stimulate the defence mechanism, and it must be complex enough to interact with the various immune system components required to induce the immune response [7]. Immunogenicity is the ability to stimulate the production of specific antibodies, which can be improved by increasing the molecular weight of small antigenic peptides, either by conjugating them with bulky proteins such as keyhole limpet haemocyanin or by increasing the number of antigenic epitopes [7,8]. In this article, the epitope approach was used by designing a multiple antigenic peptide (MAP) dendrimer because the selected antigenic region of exosomes tetraspanins was small and not enough to be an immunogen. MAP is considered to be a better immunogen than the traditional method of peptide conjugation with bulky carrier protein for making it immunogenic, and it is a relatively simple and quick process [9]. Moreover, MAPs are designed to pack multiple copies of the selected immunogenic epitope in a dense format with a high molar ratio that provides a strong immunological response [10]. It comprises several copies of small peptides attached in a core matrix of lysine residue to form an organized cascade of a branched macromolecule. There are many amino acids used as a core matrix in MAP formation, but lysine is preferred due to its epsilon amino group, which gives the protein flexibility and allows it to avoid steric hindrance [10]. The size of MAP macromolecules, which are typically 4–16 branched in length, is determined by the length of the peptide to be conjugated [11]. Over the last few years, MAPs have been used dynamically in a variety of fields such as protein mimetics, magnetic resonance imaging, peptide dendrimers, and bacterial parasitic and viral vaccine development to improve immunogenicity and potency while also investigating additional appliances [12]. The primary advantage of MAP is its resistance to enzymatic degradation and flexibility in incorporating peptides in various structural branches, which aids in the loading of antigenic peptides [12].

In this study, chickens were used for antibody production because they are a low-contamination, non-invasive method of producing antibodies that can be used in immunoprophylaxis, immunodiagnosis, and immunotherapy [13]. In poultry, serum immunoglobulin Y is transferred in the egg yolk while the egg is in the ovary, and immunoglobulins IgM and IgA are diverted in the albumen along with oviduct secretions during the egg’s passage through the oviduct [13]. Chickens produce more antibodies than other animals, with IgY isolates ranging between 100 and 400 mg per egg [13]. IgY is more suitable for diagnostic purposes than mammalian antibodies due to structural differences and phylogenetic distance, as it does not interact with certain components of the human immune system and has a greater affinity for mammalian-conserved proteins [14]. IgY antibodies have advantages such as improved animal welfare, increased productivity, and stability [15]. Furthermore, according to one study, IgY antibodies are highly stable in aqueous conditions at pH 4–9 up to 65°C, and their antigen-binding activity remains consistent in the presence of pepsin at pH 4–6 [16]. Chicken IgY antibodies contain higher carbohydrate content as compared to IgG antibody, which helps to enhance the binding of antibodies to the surface and also provide more space for conjugating with other molecules such as enzymes, peptides, and nanoparticles [17]. In addition, some researchers discovered that IgY batches have varying efficacy and cannot be purified by protein A or G, making them unsuitable for therapeutic purposes [15,18]. However, this is entirely dependent on the study’s purpose.

Antigen was designed as a MAP of the selected immunogenic peptides of CD9, CD63, and CD81 arranged in a cascade of lysine core matrix for antibody production and then immunized in chickens. Chicken IgY antibodies contain higher carbohydrate content as compared to IgG antibody which helps to enhance the binding of antibodies to the surface and also provide more space for conjugating with other molecules such as enzymes, peptides, and nanoparticles [17]. The study aimed to develop an in-house ELISA for the detection of exosomes from various biological fluids including cattle blood, cattle plasma, human urine, human serum, cell line supernatant, and clarified milk. Exosomes can be easily detected using this method for disease diagnoses, such as cancer, neurodegenerative diseases, heart failure, and liver diseases. Furthermore, these exosomes can also be used for miRNA extraction, which is believed to be highly specific in various cancerous conditions [19]. This technique may be useful for monitoring the patient’s clinical and biological conditions in various pathology conditions.

2 Materials and methods

2.1 In silico prediction of tetraspanin epitope

The antibody response is entirely reliant on specificity, and several structural epitopes of an antigen play a significant role [20]. These epitopes in the antigenic protein are formed by various continuous or discontinuous amino acid strands that must be in close spatial proximity to function properly. However, it is not necessary to select the entire protein sequence to produce target antibodies; rather, such an immunogenic portion can be selected to boost the immune system. To predict such immunogenic portions of exosome surface tetraspanins (CD9, CD63, and CD81), the protein sequences were first retrieved from the NCBI database. The Protean™ system (DNAStar, Inc., USA) was used to select the parameters of the protein’s highest antigenic region, which included hydrophilicity, accessibility, flexibility, and antigenicity [21]. Tetraspanin regions with high surface accessibility and flexibility, good hydrophilicity, and a high antigenic index were chosen as suitable epitopes for raising anti-CD9, CD63, and CD81 antibodies.

2.2 Designing and synthesis of MAP

A MAP dendrimer comprises a core matrix of amino acids, primarily lysine, which gives the structure flexibility due to the alpha (α) and epsilon (ε) amino groups. The antigenic peptide bound to these α and ε amino groups of the non-antigenic lysine provides stability from enzymatic degradation and elicits a better immune response compared to single peptides [22]. CSBio^® Ltd. synthesized the MAPs for three tetraspanins (CD9, CD63, and CD81), and the constructed backbones of the conjugated peptide sequence are disclosed in Figure 2.

2.3 Immunization of chickens

Three MAP scaffolds synthesized for tetraspanins were administered intramuscularly every 14 days for 8 weeks to an 8-month-old white leghorn bird. On the day of immunization, 100 μg of MAP protein was dissolved in 150 μL of PBS and 350 μL of Montanide adjuvant (Seppic Inc., USA) and administered to each bird. The bird administered only with PBS and adjuvant was considered as a control. The addition of adjuvants enhanced the response of the immune system against the antigen by stimulating the B-cell response [23,24]. Following immunization, the eggs were collected for IgY antibody purification, and immunoreactivity was assessed using the ELISA method.

2.4 Antibody purification

The antibodies were extracted from chicken egg yolks using the salting-out phenomenon of proteins [24,25]. In a nutshell, egg yolk from immunized chicken eggs was diluted in ten volumes of deionized water and homogenized to form a uniform suspension. The pH of the above mixture was then adjusted to 5.0 using 0.1 N HCl before being stored at −20°C overnight. The frozen sample was thawed at room temperature the next day without shaking in order to precipitate all of the lipids and phospholipids while leaving the protein in the supernatant. The supernatant was then collected by filtration and 8.8% NaCl was added to it. The pH of the above mixture was then adjusted to 4.0 with 0.1 N HCl and incubated at room temperature for 1 h. Following incubation, the precipitated protein containing IgY antibodies was collected by centrifugation at 5,000 rpm for 10 min and resuspended in an appropriate volume of PBS. The protein concentration was determined using the UV absorbance method and stored at 4°C for future use.

2.5 SDS-PAGE

Based on the molecular weight and number of bands observed, the purity of the isolated IgY samples was determined using 12% SDS-PAGE according to the Laemmli method [26]. A protein ladder with a range of 20–95 kDa (SRL Chemicals) was used as a molecular weight marker. The gel was run at room temperature for 45 min at 200 V, and bands were visualized using Coomassie brilliant blue stain.

2.6 Reactivity of IgY antibodies by ELISA

The IgY antibodies were titrated, and the reactivity of the antibodies in biological fluids was assessed using indirect-ELISA. In brief, antigen coating was performed in flat bottom microtiter plates (Thermo Scientific, USA) at a concentration of 500 ng‧well⁻¹ in carbonate/bicarbonate buffer (100 mM sodium carbonate, 100 mM sodium bicarbonate, pH 9.5), followed by a 1 h incubation at 37°C. Following incubation, the plates were washed three times with PBST (PBS containing 0.05% Tween 20), and 300 μL of the blocking buffer (5% skimmed milk prepared in PBST) was added. For dilutions of primary antibodies in diluent buffer (1/10 dilution of blocking buffer in PBS), a 10 µg solution was prepared, and two-fold diluted until the seventh well of the plate, while the last well was left empty and only diluent buffer was added in it. Plates were incubated for 1 h at 37℃ and washed again. The HRP-conjugated secondary antibodies (anti-chicken antibodies) were then diluted in the same diluents buffer at a 1:6,000 ratio and added to the plate, followed by 1 h of incubation at 37°C and washing. Finally, for colour development, the substrate buffer (100 μg‧mL⁻¹ of tetramethylbenzidine, 0.01% H₂O₂ in 50 mM citrate buffer, pH 5.0) was added to each well and incubated for 20 min at room temperature before adding 50 µL of stop solution (1 N H₂SO₄).

In the case of Sandwich-ELISA, capturing antibodies were used as 10 ng‧well⁻¹ followed by the crude biological fluids containing exosomes as an antigen source. This was further detected with anti-CD81 HRP-conjugated antibodies at a dilution of 1:2,000. A recent article technique was used to internally conjugate the anti-chicken HRP secondary antibody and anti-CD81 HRP labelled antibody, yielding a titre of 1:6,000 and 1:2,000, respectively [27]. By using the FLUOstar Omega spectrophotometer (BMG LABTECH’s), the optical density was measured at 450 nm.

3 Results

3.1 Immunogenic region of exosomal surface tetraspanins

The CD9 gene encodes a 228 amino acid transmembrane protein with four transmembrane regions, two extracellular loops, and two intracellular loops. The extracellular loops are rich in several disulphide bonds which are conserved throughout the tetraspanin family, and palmitoylation sites help in interaction with several proteins and lipids. Using an in silico prediction tool, the portion of extracellular loops conferring high antigenicity, hydrophilicity, and flexibility was chosen for anti-exosomal antibody production.

The chosen peptide was 20 amino acid long, commencing at the 124th amino acid and ending at the 143rd amino acid of the CD9 sequence (Figure 1). Similarly, peptide sites with a high antigenic index were selected for the other tetraspanin proteins CD63 and CD81, which were 238 and 236 amino acid long, respectively. For CD63, a 19mer peptide sequence was selected starting from 115th to 133rd amino acids, whereas, for CD81, a 30mer peptide sequence starting from 116th to 145th was utilized for the MAP synthesis (Table 1).

Figure 1

The structural position of folded tetraspanin CD surface receptor proteins and the selected epitope region for MAP synthesis of CD9 (a), CD63 (b), and CD81 (c).

Table 1

Amino acid sequence of the selected tetraspanin region as an immunogen and their biochemical parameters

Protein name	Position	Peptide sequence	Molecular weight (kDa)	pI	Charge
CD9	124–143 (19mer)	FYKDTYNKLKTKDEPQRETL	2.5	8.58	0.91
CD63	114–132 (19mer)	EFNNNFRQQMENYPKNNHT	2.42	7.15	0.08
CD81	115–144 (30mer)	NKDQIAKDVKQFYDQALQQAVVDDDANNAK	3.4	4.13	−2.09

3.2 Synthesis of MAP for antigenic tetraspanin region enhancement

The immunogenicity of the selected antigenic peptide from tetraspanins was achieved by artificially arranging the peptide into several branches scaffolded by a core sequence composed of Lys residues, as shown in Figure 2. A peptide assembly known as MAP is considered to improve the immunological response compared to a small peptide because the complexity and molecular weight have been increased, both of which are required for a good immunogen to awaken the immune system. It also eliminated the need for a carrier protein to elicit an antibody response.

Figure 2

Designed MAP biomolecule with the selected peptide sequence: (a) MAP of CD9, (b) MAP of CD63, and (c) MAP of CD81. (d) The molecular weight of all three constructed MAP structures.

Typically, the number of branches used to construct the MAP structure is determined by the number of amino acid residues in the peptide [28]. Based on the peptide size, a four-branched MAP dendrimer was designed for CD9 and CD63, while a two-branched MAP was designed for CD81, which is a relatively long polypeptide. It is assumed that if a long peptide is constructed in a highly branched format, it will create steric hindrance for the structure [28].

Furthermore, reverse-phase high-performance liquid chromatography (HPLC) was used to purify MAP (Figure 3). The HPLC purity of constructed MAP for CD9, CD63, and CD81 was 83.11%, 87.93%, and 82.61%, respectively, and these were suspended in PBS/DMSO in a 1:1 ratio.

Figure 3

The reverse-phase HPLC purity graph: (a) the CD9 peak, (b) the CD63 peak, and (c) the CD81 peak.

3.3 Antibody purity and titre

SDS-PAGE electrophoresis was used to determine the purity of chicken-IgY antibodies raised against the MAP antigen of exosomal tetraspanins. The purity and yield of antibodies must be optimized based on a variety of factors such as the extraction method and laboratory conditions, and purity may vary slightly as a result of these differences. The salt precipitation of antibodies was accomplished in two major steps, namely the exclusion of lipids and the precipitation of total antibodies, yielding more than 80% purity. Two prominent bands were observed in SDS-gel, indicating that one corresponded to a molecular weight of approximately 67 kDa and the other to approximately 28 kDa (Figure 4).

Figure 4

Polyacrylamide gel electrophoresis (SDS PAGE) image with bands of IgY antibodies extracted from egg yolk (Lane 1: anti-CD9 antibodies, Lane 2: anti-CD63 antibodies, Lane 3: anti-CD81 antibodies, Lane 4: protein ladder).

Anti-MAP antibody titration was monitored on a regular basis, along with each booster dose. To determine antibody kinetics, an indirect ELISA was used with 500 ng of antigen (MAP) and 10 μg of primary antibodies per well, followed by the addition of secondary antibodies (anti-chicken HRP conjugated) at a 1:6,000 ratio in each well, including a negative control with no primary antibody. The immune response of antibodies was found to be significantly increased until the third immunization and then reached a steady level, as shown in Figure 5. Indirect-ELISA was used to optimize the titration of all three antibodies, which is reliant on the net specific signal level between the background of the negative control and the sample. All three antibodies subsequently achieved a remarkable titer of approximately 1 μg of antibody, yielding a 5-fold signal difference when compared to the background Figure 5.

Figure 5

(a) A graphical representation for booster kinetics change of anti CD9, CD63, and CD81 antibodies during the immunization in chicken. (b) Illustration of anti CD9, CD63, and CD81 antibodies titration through indirect ELISA method.

3.4 Detection of exosomes in various biological fluids

Sandwich-ELISA was employed to assess the availability of exosomes in biological samples, and several combinations of antibodies that recognize CD9, CD63, and CD81 were tested. As a source of exosomes, various biological samples (cattle blood, cattle plasma, cell supernatant, cow milk, human urine, and human serum) were used in the assay (Figure 6).

Figure 6

Systematic graphical representation of exosome detection from biological fluids by Sandwich-ELISA: (a) cattle blood, (b) cattle plasma, (c) cell supernatant medium, (d) crude cattle milk, (e) human serum, and (f) human urine.

Anti-CD81 HRP labelled antibodies were used as detection antibodies in all of the assays (conc. mentioned above). The obtained results were graphed, with the best result obtained in human urine standing out.

4 Discussion

Exosomes are highly desired as a crucial tool for diagnosis and treatment in clinical and preclinical trials, where they are being studied for biodistribution, drug delivery, tracking, and tumour-targeting agents [1,29]. Regardless of the development of numerous novel technologies for exosome isolation and characterization, limitations are always a way to keep the field evolving. Exosomes secreted by the parent cell contain late endosomal components as well as various exosomal markers such as flotillin-1, heat shock proteins, major histocompatibility complex, and tetraspanins (CD6, CD9, CD63, CD81, CD82) that can be used to efficiently recover exosomes from body fluids [16]. However, the number of CD markers present on exosomes is ascertained by the biological condition of the cell, and, among them, CD9, CD63, and CD81 have been reported to be dominantly expressed, and thus, are widely used in exosomal purification [29,30]. A similar approach was used in the current study, which used immunoaffinity-based detection of exosomes via CD9, CD63, and CD81 tetraspanins. These CD markers are members of the transmembrane 4 superfamily of proteins (TM4SF), which contain four transmembrane domains and are widely used as an immunoaffinity capturing marker [31].

In the present investigation, the antibodies were preferred to be raised in chicken, which is an alternative source of mammalian antibodies and comparatively cost-effective, highly sensitive, and biochemically more active [17,23]. Instead of conjugating with the carrier protein, the antigenic region of tetraspanins was constructed using multiple epitope formation [23]. It has been reported that the highest percentage of the epitope is beneficial for epitope-specific antibodies when compared to the carrier protein; therefore, a large number of carrier-protein-specific antibodies will be generated simultaneously [22]. The MAP system has been published for the detection of HIV-specific antibodies in AIDS patients and may serve as a potential synthetic vaccine [22]. However, no quantitative approach was used to confirm that the MAP configuration improves the assay’s capacity over full-size protein or any truncated peptide sequence. In this study, the antigenic epitope of CD markers for MAP synthesis was optimized using 3D conformational analysis with the PROTEAN module of DNASTAR software [23]. Table 1 lists the designed and synthesized MAP structures, and the resulting antibodies were purified using the salt precipitation method [14]. SDS PAGE was used to confirm the purity of the antibodies, which yielded a heavy-chain band of approximately 68 kDa and a light-chain band of approximately 27 kDa, which is consistent with other studies [23,25].

The antibodies were optimized using ELISA to assess their immunogenicity for exosomes found in body fluids. Indirect ELISA and Sandwich-ELISA were used to detect antibody titration and capturing affinity. The kinetics of antibody titration revealed that the maximum affinity occurred after the third immunization and remained constant for a long time (Figure 3). Sandwich-ELISA, on the other hand, has shown that its sensitivity is highly reliant on the capturing antibodies and antigen concentration, as reported in several studies [2,32]. The cell supernatant used in this study was obtained from the Vero cell line (purchased from NCCS Pune), whereas the other biological fluids were collected locally. Furthermore, we attempted to avoid the purification step of exosomes by using crude samples as a source of exosomes because it was both quick and cost-effective, and it was also followed in previous studies [2]. Furthermore, several findings indicate that cattle are the ideal model for exosome profiling, and on that basis, cattle blood, plasma, and milk were examined to detect exosomes [33,34]. Moreover, the results show that the sensitivity of cattle plasma is approximately 50% higher than that of cattle blood, which could be attributed to the removal of excessive protein from the blood while separating plasma (Figure 4). Similarly, milk contains a high amount of protein, but milk exosome sensitivity was lower than cattle plasma but higher than cattle blood, indicating that milk contains a greater number of exosomes than blood. As previously stated, there are differences in the number of exosomes derived from different samples, and some biological contaminants may also impair assay sensitivity. Likewise, Mitchell et al. used immunoreactive CD63 antibodies to characterize exosome profiling in plasma in dairy cow strains [35]. Despite this, among all antibodies in cattle blood and plasma, CD9 appears to have the highest affinity, followed by CD63 and CD81. However, CD81 was found to be significantly higher in cow milk than CD63 and CD9. This variation was stated by the number of CD markers expressed on the surface of exosomes, which varies depending on the pathophysiological conditions of the animal [30,36].

When comparing all biological fluids, human urine exosomes were found to have the highest affinity, while cell supernatant had the lowest. Correspondingly, in another study, urine was discovered to be a good source of exosomes, with the highest number of them [37]. On the other hand, the sensitivity obtained in the Vero cell line supernatant could be attributed to the number of exosomes per mL or any interference with other secreted biomolecules, which could be improved by concentrating the exosomes with polyethylene glycol or centrifugation.

Human serum exosomes were also detected successfully using these antibodies with a good sensitivity range, indicating the presence of exosomes in human blood. As a result, this research advances the detection of exosomes. All of the body fluids used in the above study respond differently to each antibody, which could be due to animal health or the expression of a specific surface marker on exosomes. Furthermore, some effort is required to enhance this study because we used crude biological samples rather than purified samples to avoid the step of ultracentrifugation, which might boost assay sensitivity. Although there is a lot of research being done for exosome detection, the antibody raised in chickens makes it commercially viable as well as beneficial for theoretics.

5 Conclusion

The exosome detection method using ELISA in biological fluids discussed in the present study was a successful method. This demonstrated that the antibodies raised against the novel MAP design performed flawlessly in the assay. Furthermore, exosome detection via immunoaffinity would be an excellent strategy for developing exosome-based diagnostics. To avoid bulky protein conjugation, the MAP strategy would be an effective approach for eliciting antibodies against a small peptide or a recombinant immunogenic region of a protein. This technique would be used in future research, such as diagnosis or miRNA extraction, with nanoparticles conjugated with antibodies serving as not only a detection but also a separation tool for exosomes from biological fluids. miRNA is a type of circulatory molecule that is either enclosed by exosomes or mature, and it is being studied as a potential biomarker for cancer diagnosis. This study, on the other hand, provides a first step towards optimizing techniques for exosome isolation and detection with robustness and high throughput. This would help researchers working on EVs and circulating RNAs found in exosomes, which are being used as a disease diagnostic marker.

Acknowledgments

The authors are thankful to IgY Immunologix India Private Limited, Hyderabad, India, and Vignan’s Foundation for Science, Technology and Research for providing financial support and necessary facilities.

Funding information: The authors thank IgY Immunologix India Private Limited, Hyderabad, India for providing financial support.
Author contributions: Dikshita Panwar: carried out the work and writing the original draft; Lavleen Kumar Gupta: conceived the idea and designed the work; Deepali Shrivastava: contributed in laboratory techniques; Shalaka Bhawal: contributed in laboratory techniques; N. S. Sampath Kumar: contributed in review; Anjani Devi Chintagunta: responsible for review and editing.
Conflict of interest: The authors state no conflict of interest.
Data availability statement: The data generated or analysed during this study are included in this published article.

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Received: 2022-11-07

Revised: 2023-02-07

Accepted: 2023-03-09

Published Online: 2023-05-12

This work is licensed under the Creative Commons Attribution 4.0 International License.

Articles in the same Issue

https://doi.org/10.1515/revac-2023-0056

Keywords for this article

exosomes; ELISA; immunoaffinity; multiple antigenic peptides; tetraspanins

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