Jing Lin, PhD, Ludmilla Bardina, MSc, Wayne G

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Presentation transcript:

Development of a novel peptide microarray for large-scale epitope mapping of food allergens Jing Lin, PhD, Ludmilla Bardina, MSc, Wayne G. Shreffler, MD, PhD, Doerthe A. Andreae, MD, Yongchao Ge, PhD, Julie Wang, MD, Francesca M. Bruni, MD, Zhiyan Fu, PhD, Youngshin Han, PhD, Hugh A. Sampson, MD Journal of Allergy and Clinical Immunology Volume 124, Issue 2, Pages 315-322.e3 (August 2009) DOI: 10.1016/j.jaci.2009.05.024 Copyright © 2009 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 1 Time course experiment for optimization of serum incubation. Three replicate arrays were immunolabeled with 1/500 diluted serum pool or 1/5 diluted negative control serum for each condition. Data presented are averaged z scores of the replicates. Reactions to the pool and negative control serum are showed as ♦ and □, respectively. The percentages of positive peptides (z score>3) are indicated. Journal of Allergy and Clinical Immunology 2009 124, 315-322.e3DOI: (10.1016/j.jaci.2009.05.024) Copyright © 2009 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 2 Scatter plots showing the effects of different variables (printing lot, immunolabeling day, and serum pool batch) on reproducibility of the peptide microarray. A, Data from 2 replicate arrays with the same 3 variables. B-D, Data from 2 replicate arrays differing in 1 of the 3 variables: immunolabeling day (B), serum pool batch (C), or printing lot (D). Coefficient of correlation was calculated (P < .01) and shown in each scatter plot. Journal of Allergy and Clinical Immunology 2009 124, 315-322.e3DOI: (10.1016/j.jaci.2009.05.024) Copyright © 2009 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 3 Results from serum dilution experiment (A) and peptide inhibition assay (B). Data are presented as Z scores by using TMeV. Areas showing non-specific binding are indicated with ◀. Targeted peptides, which are the peptides pre-incubated with the serum pool are indicated with black arrows for each inhibition group. Areas with possible cross-inhibition based on sequence alignment are indicated with grey arrows. Journal of Allergy and Clinical Immunology 2009 124, 315-322.e3DOI: (10.1016/j.jaci.2009.05.024) Copyright © 2009 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 4 Comparison between the epitopes identified from SPOT membrane immunoassays and peptide microarray. Peptides with amino acid sequences of at least 90% overlapped with the major epitopes and minor epitopes identified using SPOT membrane technology are shown in and , respectively, whereas peptides with positive reactions (z score >3) to positive serum pool in peptide microarray are shown in . Journal of Allergy and Clinical Immunology 2009 124, 315-322.e3DOI: (10.1016/j.jaci.2009.05.024) Copyright © 2009 American Academy of Allergy, Asthma & Immunology Terms and Conditions

An experiment of pin printing consistency showing the fluorescent intensity (DFU, spot median-local background) of 288 spots of 1.5 μg/mL Alexa 546–BSA in PPB printed from each of the 8 spotting pins in a row without reloading. Journal of Allergy and Clinical Immunology 2009 124, 315-322.e3DOI: (10.1016/j.jaci.2009.05.024) Copyright © 2009 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Test of variable degrees of humidity showing the fluorescent intensity (DFU) of 80 spots of 1.5 μg/mL Alexa 546–BSA printed in various humidity conditions ranging from 35% to 62%. Journal of Allergy and Clinical Immunology 2009 124, 315-322.e3DOI: (10.1016/j.jaci.2009.05.024) Copyright © 2009 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Triplicate spots of 5 peptide samples on slides labeled with the 1/5 diluted serum pool. The triplicate spots of Alexa 546–BSA (1.5 μg/mL in PPB) were also shown as a comparison. Peptides were printed as 0.33 mg/mL in PPB, 33% DMSO (A); 0.33 mg/mL in PPB, 33% DMSO, 0.02% Sarkosyl (B); and 0.50 mg/mL in PPB, 50% DMSO, 0.02% Sarkosyl (C). Journal of Allergy and Clinical Immunology 2009 124, 315-322.e3DOI: (10.1016/j.jaci.2009.05.024) Copyright © 2009 American Academy of Allergy, Asthma & Immunology Terms and Conditions