## 3 Results
### Optimization of Exosome Isolation Conditions from Various Biological Specimens  
To establish optimized protocols for exosome isolation from urine, plasma, and serum samples, we evaluated various conditions, including the magnetic bead concentration, incubation time, washing procedure, and elution duration, based on the levels of key exosomal markers (Figure 6).  
For urine samples, marker levels in samples prepared using different bead concentrations and incubation times were compared. Compared with the 2 mg bead condition, the 12 mg bead condition resulted in significantly higher levels of all exosomal markers, including THP, TSG101, CD9, ALLX, CD63, and CD81. Extending the incubation time from 10 to 20 min further enhanced marker levels. Notably, TSG101 levels were lowest for the 2 mg bead condition but reached levels comparable to those obtained by the conventional PEG method under the 12 mg/20 min condition. Additionally, omitting the washing step consistently increased the expression of all markers compared to the conventional method. Therefore, the optimal conditions for urine-derived exosome isolation were defined as follows: a bead concentration of 12 mg/mL (1.2 mL), representing a 1.2-fold increase relative  
Figure 5: Comparison of exosome isolation efficiency using EGCG-modified magnetic bead with different concentrations of EGCG and beads.  
to the sample volume, a bead binding time of 10 min, omission of the washing process, and a 20 min elution time.  
For plasma samples, we assessed exosomal marker levels in samples prepared using bead binding times of 5, 10, and 20 min. The Western blot analysis indicated high ALB (50.66 kDa) levels using the PEG method, whereas the EGCG-modified magnetic bead method resulted in significantly lower ALB levels, suggesting a reduced co-isolation of non-specific proteins such as albumin. Tetraspanin markers (CD9 at 22 kDa, CD63 at 35-60 kDa, and CD81 at 22-24 kDa) exhibited optimal levels with a 10 min bead binding time, with CD63 and CD81 levels surpassing those obtained using the PEG method. Internal markers such as ALIX (95 kDa) and TSG101 (47.52 kDa) showed increased levels with extended binding times, achieving greater levels than the PEG method with the 20 min binding condition. Based on these results, the optimal conditions for plasma-derived exosome isolation were determined as a bead binding time of 30 min with a 10 min elution period.  
Similarly, serum samples were analyzed at bead binding times of 10, 20, and 30 min. CD63 (35-60 kDa) displayed the highest levels using the PEG method but showed increased levels with longer bead binding durations when using the magnetic beads. CD81 (22-24 kDa) reached levels comparable to those using the PEG method with the 30 min binding condition. ALIX (96 kDa) levels were highest with a 10 min binding time, while TSG101 (47.52 kDa) maintained consistent levels regardless of the binding duration. Conversely, CD9 (22 kDa) levels slightly decreased with prolonged incubation. Comparing elution durations, a 10 min elution consistently yielded the optimal results. Therefore, for serum-derived exosome isolation, the ideal conditions were defined as a bead binding time of 30 min combined with a 10 min elution period.  
A Western blot analysis of exosomal markers from (A) urine, (B) plasma, and (C) serum samples isolated using PEG precipitation (PEG) and EGCG-modified magnetic beads under varying conditions was performed. The molecular weights (kDa) are shown on the left. The optimal isolation conditions were determined based on marker band intensity.