Fluorescence quenching-based immunological probe for ticagrelor monitoring

FIGURE 1. Antibody expression and activity assay. (A) Schematic structure of MEDI2452, IgG 72, IgG 152, and IgG 162; (B) SDS-PAGE coomassie brilliant blue after antibody expression purification; (C) Structure of ticagrelor-NHS ester; (D) SDS-PAGE coomassie brilliant blue of ticagrelor-NHS ester labeled with BSA after purification; (E) Mean fluorescence intensity of FACS of PAC1 to detect the inhibitory effect of BSA-Tic on platelets; (F) ELISA to detect the binding activity of antibody to BSA-Tic. Adjusted p-values in Panels (E,F) were calculated by one-way ANOVA with Tukey’s multiple comparisons test. H: antibody heavy chain fragment; L: antibody light chain fragment; Error bars, ±1 S.D (n = 3); ***: p < 0.001.

FIGURE 2. Preparation of Anti-ticagrelor Q-body and its activity detection. (A) Schematic diagram of ATTO/TAMRA-labeled antibody; (B) SDS-PAGE coomassie brilliant blue of fluorescently labeled anti-ticagrelor Q-body; (C) Fluorescence imaging of ATTO-labeled antibody; (D) Fluorescence imaging of TAMRA-labeled antibody; (E) ELISA detection of BSA-Tic binding activity of fluorescently labeled Q-body. Adjusted p-values in Panel (E) were calculated by one-way ANOVA with Tukey’s multiple comparisons test. H: Antibody heavy chain fragment; L: Antibody light chain fragment; Error bars, ±1 S.D (n = 3); ***: p < 0.001.

FIGURE 3. GdnHCl/DTT denaturation assay of anti-ticagrelor Q-body and detection of ticagrelor and digoxin in PBS. (A) Schematic diagram of Q-body detection of ticagrelor; (B) 2452A Q-body denaturation assay spectrum in GdnHCl/DTT denaturant; (C) 152T Q-body denaturation assay spectrum in GdnHCl/DTT denaturant. (D) Time-stability curves of 2452A Q-body for the detection of ticagrelor in PBS and serum; (E) Time-stability curves of 152T Q-body for the detection of ticagrelor in PBS and serum; (F) Spectra of 2452A Q-body after addition of gradient-diluted ticagrelor with PBS as background; (G) Stoichiometric-response curves for detection of ticagrelor and digoxin in PBS by 2452A Q-body; (H) Spectra of 152T Q-body after addition of gradient-diluted ticagrelor with PBS as background; (I) Stoichiometric-response curves for detection of ticagrelor and Digoxin in PBS by 152T Q-body. F.I: fluorescence intensity; GdnHCl/DTT: 7 M guanidine hydrochloride, 100 mM dithiothreitol; Error bars, ±1 S.D (n = 3).

FIGURE 4. 2452A and 152T Q-body detection of TAM and TIM in PBS and ticagrelor in spiked serum. (A) Spectra of 2452A Q-body after addition of gradient-diluted TAM with PBS as background; (B) Stoichiometric-response curves for detection of TAM and TIM in PBS by 2452A Q-body; (C) Spectra of 152T Q-body after addition of gradient-diluted TAM with PBS as background; (D) Stoichiometric-response curves for detection of TAM and TIM in PBS by 152T Q-body; (E) Spectra of 2452A Q-body after addition of a gradient dilution of ticagrelor with spiked serum as background; (F) 2452A Q-body assay stoichiometry-response curve for ticagrelor in spiked serum; (G) Spectra of 152T Q-body after addition of a gradient dilution of ticagrelor with spiked serum as background; (H) 152T Q-body assay stoichiometric response curve for ticagrelor in spiked serum. F.I: fluorescence intensity; Error bars, ±1 S.D (n = 3).

FIGURE 5. 2452A and 152T Q-body detection of TAM, TIM, and the mixture of ticagrelor and TAM (3:1) in spiked serum. (A) Spectra of 2452A Q-body after addition of gradient-diluted TAM with spiked serum as background; (B) 2452A Q-body assay stoichiometry-response curve for TAM and TIM in spiked serum; (C) Spectra of 152T Q-body after addition of gradient-diluted TAM with spiked serum as background; (D) 152T Q-body assay stoichiometry-response curve for TAM and TIM in spiked serum; (E) Spectra of 2452A Q-body after addition of gradient diluted ticagrelor: TAM (3:1) with spiked serum as background; (F) Stoichiometric response curve of 2452A Q-body for detection of ticagrelor: TAM (3:1) in spiked serum; (G) Spectra of 152T Q-body after addition of a gradient dilution of ticagrelor: TAM (3:1) with spiked serum as background; (H) Stoichiometric response curve of 152T Q-body for detection of ticagrelor: TAM (3:1) in spiked serum. F.I: Fluorescence intensity; Error bars, ±1 S.D (n = 3).