Fig 2: Quantitative Detection in ALPPL2 or CD9 Antibody/SQ2 Aptamer Sandwich ALISA(A) Scheme of sandwich ALISA. (B) ALPPL2 antibody/SQ2 aptamer-based sandwich ALISA for detecting recombinant ALPPL2 proteins with sensitivity of 3.5 ng (= 35 ng/mL) of ALPPL2. (C) The standard curve showed linearity in the range from 5 to 500 ng/mL. (D) ALPPL2 antibody/SQ2-based detection of ALPPL2 in PANC-1+ EVs (E) CD9 antibody/SQ2 aptamer-based sandwich ALISA for detecting PANC-1+ EVs. Sensitivity of detection for PANC-1+ EVs was as low as 0.1 ng (= 1 ng/mL). (F) CD9 antibody/SQ2 sandwich ALISA for detecting ALPPL2 in three PDAC-derived EVs (5 μg/mL) showed signals comparable to direct ALISA.

Fig 3: Proof-of-Principle Liquid Biopsy Test Using ALPPL2 Negative Human Serum Spiked with PANC-1+ EVs in CD9 Antibody/SQ2 Aptamer-Based Sandwich ALISASerum was diluted in a ratio of 1:1 to 1:100 in PBS and then spiked with 0.5 μg of EVs. Samples were directly tested or EVs were isolated by Exoquick-based precipitation method and then tested with this sandwich ALISA.

Fig 4: Aptamer SQ2-Based Direct ALISA for Quantitative Analysis of PDAC-Derived EVs(A) Schematic illustration of SQ2 aptamer-based direct ALISA for EV detection. (B) SQ2-based ALISA can detect recombinant ALPPL2 protein with a sensitivity of 1 ng (10 ng/mL). (C) Standard curve showing linearity in the broad range of 10 to 2,500 ng/mL of protein. (D) ALPPL2 estimation in the secretomes and EVs of (D) PANC-1+, (E) Capan-1, and (F) MIA PaCa-2 cells using SQ2-based ALISA. ALISA could detect ALPPL2 in EVs with much higher sensitivity than in the secretome. Results are mean ± SD of more than three independent experiments.