Figure 1.

ORF1p expression is early and pervasive in carcinomas. a, ORF1p immunostaining in a cohort of 211 colorectal cancers. b, Representative BE case: lesional cells overexpress p53, the L1 RNA, and ORF1p. c, L1 RNA and ORF1p overexpression across a cohort of 72 consensus BE cases and 51 carcinomas. d, Schematic of single-molecule protein detection by Simoa; a second generation assay is shown. Antibody/nanobody-coated magnetic beads, present in excess relative to target, capture single target ORF1p molecules. Enzyme-labeled detection reagent (here, a homodimeric nanobody) is added, forming an “immunosandwich”, beads are loaded into microwells that each can hold at most one bead, and ORF1p molecules are then digitally detected using a fluorogenic substrate by counting “on” wells. First generation Simoa instead uses Nb5-coated beads and Ab6 detector e, First-generation ORF1p Simoa detects plasma ORF1p with high specificity across major carcinomas. Pie charts indicate percentage of samples with detectable levels; dashed red line, LOD. **, this control patient was thought to be ‘healthy’ at the time blood was donated to the biobank but was later found to have prostate cancer and lymphoma.