Fig 1: 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 2: ALPPL2 Is Present in PDAC Cell-Derived EVsImmunoblot analysis of EVs extracted from the serum-free conditioned media of PANC-1+ and Capan-1 showed significant ALPPL2 expression, whereas MIA PaCa-2 showed no expression of the protein. 15 µg of secretome (S), 15 µg of EV-depleted secretome (X), and 2.5 µg EVs (E) were used for immunoblotting. EV markers CD9, CD63, and TSG101 confirm the presence of EVs in samples S and E.
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.
Supplier Page from Aviva Systems Biology for CD9 ELISA Kit (Human) (OKCD00751)
Specificity: This assay has high sensitivity and excellent specificity for detection of Motility Related Protein (MRP1). No significant cross-reactivity or interference between Motility Related Protein (MRP1) and analogues was observed.