*2.7. YGS Treatment Shows Reduced Aβ Expression for Aβ<sup>42</sup> Flies by IMR Assay*

We used a highly sensitive IMR assay that can detect ultralow concentrations of Aβ protein in human blood for early diagnosis of AD [23–25]. We established a curve of the relationship between Aβ<sup>42</sup> expression and the number of GFP-Aβ<sup>42</sup> flies by using an IMR assay. Our results showed that the increase in the IMR signal as Aβ<sup>42</sup> concentration increased was linked to the number of Aβ42-expressing flies (Figure 6A, Right). In addition, we plotted a relationship curve and then found that the relationship between IMR signals

and Aβ<sup>42</sup> expression is positively correlated (Figure 6A, Left). IMR signals (in percentages) for Aβ<sup>42</sup> concentrations between 0.1 and 10,000 pg/mL were used to explore the analytical relationship, which followed a logistic function. According to the relationship curve of Figure 6A, we quantified and then compared that the Aβ<sup>42</sup> concentration of GFP-Aβ<sup>42</sup> flies with sham treatment was significantly stronger than those of GFP-Aβ<sup>42</sup> flies with 1% YGS treatment (sham, 26.4 pg/mL vs. YGS, 22.8 pg/mL, *p* < 0.05, Figure 6B). The results revealed down-regulation of Aβ<sup>42</sup> expression for GFP-Aβ<sup>42</sup> flies under YGS treatment.

**Figure 6.** IMR assay of Aβ<sup>42</sup> expressions for Aβ42-GFP flies under sham and YGS treatments. (**A**) Left shows a relationship between Aβ<sup>42</sup> concentrations and the number of Aβ42-GFP flies by IMR assay. A magnetic nanoparticle coated with Aβ<sup>42</sup> bioprobes was shown. Right shows the IMR value was increased with the Aβ<sup>42</sup> concentration. (**B**) Quantified Aβ<sup>42</sup> concentration of Aβ42-GFP flies under sham treatment was significantly greater than those of Aβ42-GFP flies under YGS treatment (*N* = 100 for each group). Values are mean ± SEM (\* *p* < 0.05, one-way ANOVA followed by a Student– Neman–Keuls multiple comparisons post-test). Abbreviations: YGS, Yi-Gan-San; Aβ, amyloid-beta, GFP, green fluorescent protein; IMR, immunomagnetic reduction; SEM, standard error of the mean; ANOVA, analysis of variance.
