Journal: Advanced Science

Article Title: Enzyme‐Linked Cycloaddition Assay (ELCA) for Rapid, Ultra‐Sensitive Monitoring of Secreted Sialoglycoproteins

doi: 10.1002/advs.78315

Figure Lengend Snippet: Quantifying secreted glycoproteins under standard culture conditions. (A) Schematic of the ELCA workflow. Supernatant samples containing azide‐modified glycoproteins are collected from metabolically labeled cells, adsorbed onto microwell plates, and subsequently labeled via strain‐promoted cycloaddition. Detection and quantification are performed using HRP. (B) XTT assay quantifying cellular metabolic activity under culture conditions with varying serum concentrations. ( n = 8). (C) ELCA analysis of supernatants from cells cultured under varying serum concentrations using standard colorimetric detection ( n = 4). (D, E) Signal‐to‐noise ratio (S/N) of ELCA signal comparing standard culture conditions (DMEM + 10% serum; (D) and serum‐free Opti‐MEM (E) at 24 h and 72 h time points, measured by standard colorimetric detection ( n = 4). (F) S/N of ELCA signal from cells cultured under standard conditions at 24 h and 72 h time points, measured using high‐sensitivity luminescent detection ( n = 4). (G, H) ELCA signal following treatment with 3 µ m Brefeldin A (BFA) for 24 h under serum‐free (G) or standard culture conditions (H), measured using high‐sensitivity luminescent detection ( n = 4). – (gray bars): blank control. (I–K) ELCA measurements of total glycoprotein secretion (I), Sandwich‐ELCA measurements of glycosylated GM‐CSF secretion (J), and ELISA measurements of total GM‐CSF secretion (K) following PMA stimulation in cells cultured under standard conditions for 6–48 h, using high‐sensitivity luminescent detection (I; n = 3) or standard colorimetric detection (J–K; n = 3). All assays were conducted in A375 cells. Statistical significance was assessed using a two‐tailed Welch's t ‐test (two conditions), one‐way ANOVA with Dunnett's multiple comparisons test (>2 conditions, 1 variable), or two‐way ANOVA with Tukey's multiple comparisons test (<2 conditions, 2 variables). * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: For detection, the plate was incubated with 0.5 μg/mL GM‐CSF polyclonal antibody (17762‐1‐AP, Proteintech) for 1 h at room temperature followed by 40 ng/mL HRP‐conjugated Goat Anti‐Rabbit IgG (SA00001‐2, Proteintech) for 1 h at room temperature.

Techniques: Modification, Metabolic Labelling, Labeling, XTT Assay, Activity Assay, Analysis, Cell Culture, Sensitivity, Control, Enzyme-linked Immunosorbent Assay, Two Tailed Test