**6.3 ANS fluorescence analysis**

1-anilinonaphthalene-8-sulfonate (ANS) is one of the most commonly used fluorescent probes for characterization. ANS provides an assessment of surface hydrophobicity, depicting increase in fluorescence intensity and a blue shift (decrease in wavelength) when exposed to hydrophobic regions of the protein surface [73]. The interaction and subsequent destruction of the bilayer of the hydrophobic lipid membrane is considered as one of the main mechanisms conferring toxicity to cells in diseases involving prefibrillar aggregation. Therefore, assessment of the surface hydrophobicity of protein aggregates could potentially be very useful for the study of fibrillar protein aggregates [74]. Indeed, a recent study of prefibrillar oligomers of Aβ42 peptide showed an increase in fluorescence and a

change in blue color upon exposure to the ANS compared to fibrils and monomers, as well as a correlation between increased ANS fluorescence and toxicity. In certain areas of monitoring prefibrillar protein aggregates, ANS is used less frequently than CR and ThT [72, 75].

#### **6.4 Antibody dot blot assay**

Due to the difficulty to obtain high-resolution crystal structures of protein aggregates (especially fibrillar aggregates), structure-specific antibodies that help identify and control the state of amyloidogenic protein aggregates have been developed in the past decade. In a study by Glabe [76] have developed three conformation-specific antibodies important for the detection of physiologically relevant fibrillar aggregates: A11 (recognizing fibrillar oligomers but not fibrillar conformers) and OC (fibrillar oligomers, fibrillar conformers) [77]. These conformation-specific antibodies have the inhibitory ability of Aβ aggregation modulators, inhibition of toxic A11-reactive Aβ aggregation formation by diamond blue G (BBG), and low molecular weight inhibitor [78]. Although the application of fibrillar protein aggregates has provided an important understanding of the properties of fibrillar protein aggregates and the effectiveness of potential therapeutics, recent studies suggest caution should be exercised in the use and interpretation of results. First, due to the transient nature of the pre-fibrillar aggregates compared to the final-state conformers, it is very difficult to prepare homogeneous samples of pre-fibrillar aggregates that react exclusively with A11, OC, or αAPF (no cross-reactivity) *in vitro* [79]. It has proven difficult. Preparation of homogeneous prefibrillar aggregates. Second, when testing the inhibitory/modulatory activity of foreign compounds on protein aggregates in several study groups, false-positive antibody reactivity was observed in some cases. Because of these two factors, care must be taken when designing experiments and interpreting the results of these antibodies [72].

Direct observation of amyloid plaques *in vivo* is also used as a diagnostic tool for protein aggregation. Although this direct observation is attractive for clinical use, it is not routinely practiced. Technologies such as ELISA, magnetic resonance imaging (MRI), positron emission tomography (PET), and diffusion tensor imaging are being developed for the direct diagnosis of amyloid plaques based on visual inspection of enhanced images [80, 81]. However, all these methods are based solely on a qualitative approach and rely on the detection of visible changes in the central nervous system. Although some work has been done to quantify amyloid load in PET image analysis, the results are very limited. More recent advances in MRI are primarily based on the use of nanoparticles to localize plaques [82, 83]. For example, the use of magnetic nanoparticles bound to curcumin or hollow manganese oxide nanoparticles bound to specific antibodies. These two nanoparticle methods increase the specificity and sensitivity of the method to protein aggregates. However, these approaches are not routinely used and may not meet the need for diagnosis before irreversible tissue damage occurs [68].
