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In addition, we also discovered that known proteins with weak or no correlation to a disease exhibited a
strong correlation to disease when associated with a specific EV type of the biological fluid. Therefore, to
facilitate the discovery of candidate biomarkers and their subsequent validation in population, we routinely
screened known and well-characterized proteins for candidacy as biomarkers using antibody arrays or
[33]
ELISAs . The rationale for this strategy was two-fold. Firstly, the candidate biomarker would have a well
characterised ELISA. Secondly, the isolation technique could be readily coupled to the standard 96 well
[33]
ELISA platform leading to a semi-automated and moderately high throughput procedure .
As a proof of concept, we tested our strategy in a study to identify predictive biomarkers for pre-eclampsia
[36]
using a prospective biobank . Known and well-characterised proteins were first screened as potential pre-
eclampsia biomarkers in plasma, plasma CTB-binding EV and plasma AV-binding EV from patients. Three
different candidate biomarkers were identified, namely placental growth factor (PlGF), Tissue inhibitor of
metalloproteinase & Plasminogen activator inhibitor-1 in plasma (plasma PlGF), plasma CTB-binding EV
[36]
(CTB-TIMP1) and plasma AV-binding EV (AV-PAI1), respectively . These three biomarkers were tested
in a prospective biobank of 843 pregnant women. Pre-eclampsia was predicted about 7.3 (± 2.9) weeks
before clinical diagnosis with a combined AUC of 0.96, sensitivity of 100%, specificity of 78.6%, and PPV
of 9.9%. The cut-off concentration for predicting PE was at < 1235, ≤ 300 or > 1300 and < 10,550 pg/mL
plasma for plasma PlGF, CTB-TIMP1 & AV-PAI1 respectively.

Differences in the proteome of the different EV types were also observed in cancer patients. Using a similar
strategy as in the pre-eclampsia study, it was observed that MMP9 was elevated in AV-, but not detectable
in CTB- or ST-binding EVs from the ascites fluid of serous ovarian cancer patients. However, this MMP9
[34]
elevation was not observed in AV-binding EVs from ascites fluid of liver cirrhosis patients . More
recently, a preliminary screen of plasma, plasma CTB-binding EV and plasma AV-binding EV revealed the
presence of potential biomarkers that could prospectively predict the outcome of chemoradiation therapy
[37]
in patients with head and neck squamous-cell carcinoma .

The presence of EVs in biological fluids with different affinities for membrane lipid-binding ligands
demonstrated that the presence of concentrated, exposed domains of specific membrane lipids in some EV
types are not unique to EVs from cell cultures. Importantly, this is a physiological phenomenon and not
a consequence of the unique nutrient composition of the culture medium. It is also notable that each EV
subtypes have different proteomes and that the proteome of these EV subtypes is differentially enriched in
disease biomarkers.

ADVANTAGES AND DISADVANTAGES OF USING MEMBRANE LIPID BINDING MOLECULES
IN EV ISOLATION FOR DIAGNOSIS
There are several advantages of using membrane lipid binding molecules to isolate EVs for diagnosis. The
most important advantage is that the principle for this isolation technique is based on the defining feature
of EVs, namely the presence of a lipid membrane. Membrane lipids are generally amphiphilic, i.e., having
both hydrophobic and hydrophilic ends, and in aqueous solution, they will form micelles or liposomes,
i.e., vesicles. Therefore, it is likely that these membrane lipids especially when present in localized high
concentrations in aqueous biological fluids, are likely to be minimally in a micelle configuration, and most
possibly, a vesicular structure. The presence of membrane or cytosolic proteins will strongly corroborate
the presence of membrane vesicles.

Based on affinity for membrane lipid-binding ligands, it is obvious that biological fluids contain several
EV types with EV type-specific proteome. Importantly from the diagnostic perspective, this EV type-
specific proteome is also dependent on the pathophysiological state of the donor and this pathophysiology-