Evaluation of the Usefulness of an Automatable Immunoassay for Monitoring Celiac Disease by Quantification of Immunogenic Gluten Peptides in Urine

Figures

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  Figure 1. G12-HRP moAb titration for sandwich ELISA optimization. (A). Relative affinity of G12-HRP moAb for 33-mer peptide. (B). Standard reference curves. EC50 and cross-reactivity (CR) were obtained by G12 ELISA. EC50 is the line concentration that reduces the maximum absorbance by 50% in the assay. CR was calculated as follows: (EC50 of the antigen for which the moAb was raised/EC50 of each antigen assayed) × 100. These assays were performed in duplicate moAb, monoclonal antibody.

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  Figure 2. (A) Calculation of slopes (Δy/Δx) between the different concentrations of the curve and the correlation coefficient (r2). The slope (Δy/Δx) between concentrations was calculated using the equation; Δy/Δx = (Y1 − Y0)/(X1 − X0); Y = concentration (ng/mL 33-mer), and X = absorbance. (B). Polynomial representation of 33-mer concentrations (12.5 and 1.56 ng/mL). (C). Correlation coefficient (r2) calculated from the polynomial approximation of the standards analyzed. Abs, absorbance; CV%, coefficient of variation; SD, standard deviation.

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  Figure 3. Standard curve for the quantification of the 33-mer used as reference material. The linear portion of the curve is between 12.5 and 1.56 ng/mL. The solid black line represents the mean values and standard deviations (SD) of 12 determinations per concentration in the urine matrix and the dashed line in the dilution solution.

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  Figure 4. Stability of urinary GIP over a 16-day period. (A) room temperature. (B) 4 °C. (C) −20 °C. Volunteer one is represented in blue, two in green, and three in yellow. Each point represents the mean of duplicate measurements plus the SD if the GIP was measurable. GIP, gluten immunogenic peptides; LOQ, limit of quantification; ND, non-determined; n/a, non-applied.

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  Figure 5. Concentration of urine GIP from patients with CD at the baseline visit and over 12 months after the initiation of a GFD. (A) Percentage distribution of the GFD-treated patients with CD according to GIP concentration. (B) GIP levels in urine at the basal and follow-up visits (basal, three, six, and 12 months). CD, celiac disease; GFD, gluten-free diet; GIP, gluten immunogenic peptides; LOQ, limit of quantification.