Mediator release assay for assessment of biological potency of German cockroach allergen extracts Anna H. Nowak-Wegrzyn, MD, Ramon Bencharitiwong, BS,

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Mediator release assay for assessment of biological potency of German cockroach allergen extracts Anna H. Nowak-Wegrzyn, MD, Ramon Bencharitiwong, BS, John Schwarz, PhD, MHSc, Gloria David, PhD, MHSc, Peyton Eggleston, MD, Peter J. Gergen, MD, MPH, Andrew H. Liu, MD, Jacqueline A. Pongracic, MD, Sampson Sarpong, MD, Hugh A. Sampson, MD Journal of Allergy and Clinical Immunology Volume 123, Issue 4, Pages 949-955.e1 (April 2009) DOI: 10.1016/j.jaci.2009.01.070 Copyright © 2009 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 1 Side-by-side comparison of 3 cockroach extracts in skin tests (A) and mediator release assays (B). Data are shown for 3 representative subjects (subjects 1, 4, and 7). Dose-response curves were generated with serial dilutions of cockroach extract A (black), extract B (blue), and extract C (red). Data for all 19 responders are included in Fig E1 in this article's Online Repository at www.jacionline.org. Journal of Allergy and Clinical Immunology 2009 123, 949-955.e1DOI: (10.1016/j.jaci.2009.01.070) Copyright © 2009 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 2 Comparison of dose-response curves generated with serial dilutions of 3 cockroach extracts in 6 representative subjects (responders in the RBL assay) by skin test (A) and mediator release assay (B). Black lines represent extract A, blue lines represent extract B, and red lines represent extract C; symbols represent average values for 6 subjects. Journal of Allergy and Clinical Immunology 2009 123, 949-955.e1DOI: (10.1016/j.jaci.2009.01.070) Copyright © 2009 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 3 Dose-response curves generated with serial dilutions of 4 starting concentrations of extract E (1:2.5, 1:10, 1:25, and 1:250 wt/vol) and a 7-subject serum pool (A) and an individual subject's serum (B). NHR to cockroach extract at 10−2 dilution without serum reached a similar value to the NHR in the presence of serum; therefore this data point was not displayed. Journal of Allergy and Clinical Immunology 2009 123, 949-955.e1DOI: (10.1016/j.jaci.2009.01.070) Copyright © 2009 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 4 Results of 4 identical experiments repeated on 4 different days. Dose-response curves were generated with serial 10-fold dilutions of 3 starting concentrations of extract E (1:2.5, 1:10, and 1:25, wt/vol) and sera from 6 subjects. For subject 15 at 1:25 extract concentration, we were not able to fit the results of 3 of the experiments with the 4-parameter logistic model. Journal of Allergy and Clinical Immunology 2009 123, 949-955.e1DOI: (10.1016/j.jaci.2009.01.070) Copyright © 2009 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Side-by-side comparison of 3 cockroach extracts in skin tests (A) and mediator release assays (B). Data are shown for 19 responders. Dose-response curves were generated with serial 3-fold dilutions of 3 cockroach extracts. Black lines represent extract A, blue lines represent extract B, and red lines represent extract C. Journal of Allergy and Clinical Immunology 2009 123, 949-955.e1DOI: (10.1016/j.jaci.2009.01.070) Copyright © 2009 American Academy of Allergy, Asthma & Immunology Terms and Conditions