Fig. 2: Schematic of sandwich ELISA, the sensitivity and specificity to detect ofCSPGs.
a Schematic of sandwich ELISA workflow. The figure was created with Biorender.com. b Colocalization of ofCS with core proteins detected by rVAR2-3 and anti-CD44, CSPG4, and SDC1 antibodies (n = 3, biologically independent experiments). P = 0.0039 and P < 0.0001 for CD44 vs. CSPG4 and CD44 vs. SDC1 in A549, P = 0.0001 and P = 0.0025 for CD44 vs. CSPG4 and CD44 vs. SDC1 in SW480, P = 0.0549 and P = 0.0002 for CD44 vs. CSPG4 and CD44 vs. SDC1 in SW620. R = 1 represents perfect correlation. Scale bar = 10 μm. c The sensitivity of ELISA for ofCS-CD44, -CSPG4, and -SDC1 biomarkers. The cancer cell line (SW480) spiked samples were tested. d The ELISA specificity was detected by the competitive assay with gradient dilution of CSA (3.75 × 10−5–7.5 mg/mL), decorin, and chondroitinase-treated cancer cell line or plasma samples (n = 3, 2, and 2, respectively, biologically independent experiments). The chondroitinase treatment of the cancer cell line and patient plasma sample significantly reduced the rVAR2 binding (P = 0.0062 and P < 0.0001 respectively). No significant rVAR2 binding was detected in the decorin spiking sample indicating specific capture of the anti-CD44 antibody. Student t-test, all the tests were two-sided, data were shown as mean ± SD, ns. no significant, *p value <0.05, **p value <0.01, ***p value <0.001, ****p value <0.0001).
