Validation of an Immunoassay for Anti-thymidine Phosphorylase Antibodies in Patients with MNGIE Treated with Enzyme Replacement Therapy Michelle Levene,

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Validation of an Immunoassay for Anti-thymidine Phosphorylase Antibodies in Patients with MNGIE Treated with Enzyme Replacement Therapy Michelle Levene, Dario Pacitti, Charlotte Gasson, Jamie Hall, Marcia Sellos-Moura, Bridget E. Bax Molecular Therapy - Methods & Clinical Development Volume 11, Pages 1-8 (December 2018) DOI: 10.1016/j.omtm.2018.08.007 Copyright © 2018 The Authors Terms and Conditions

Figure 1 Specificity Cut Point To establish the specificity cut point, we pre-incubated 51 individual control serum samples with TP at a concentration of 12,500 ng/mL and analyzed them in duplicate by two analysts over three plates on 3 days to assess % signal inhibition. Significant differences were observed between the means for analyst, day, plate, analyst × day, day × plate, and analyst × day × plate interactions (p < 0.001). Data are expressed as mean % signal inhibition ± SD. Molecular Therapy - Methods & Clinical Development 2018 11, 1-8DOI: (10.1016/j.omtm.2018.08.007) Copyright © 2018 The Authors Terms and Conditions

Figure 2 Assay Tolerance to Free TP Instrument response as function of TP concentration. The LPC was spiked with TP over a concentration range of 39.1–40,000 ng/mL and incubated for 1 hr before analysis. The blue arrow indicates the assay drug tolerance. Molecular Therapy - Methods & Clinical Development 2018 11, 1-8DOI: (10.1016/j.omtm.2018.08.007) Copyright © 2018 The Authors Terms and Conditions

Figure 3 Assay Selectivity Individual control (n = 10) and disease (n = 7) serum samples were unspiked or spiked with anti-TP antibodies at low (400 ng/mL) and high (10,000 ng/mL) concentrations. Two aliquots of each sample were prepared and incubated for 1 hr, one aliquot with buffer and the other aliquot with free TP (12,500 ng/mL). Dotted line represents assay cut point. Data are expressed as log mean RLUs ± SD. Molecular Therapy - Methods & Clinical Development 2018 11, 1-8DOI: (10.1016/j.omtm.2018.08.007) Copyright © 2018 The Authors Terms and Conditions

Figure 4 Stability of Anti-TP Antibody after 24 hr at Room Temperature and after Repeated Freeze-Thaw Cycles The dashed lines represent the assay cut-point range. Data are expressed as log mean RLUs ± SD. Molecular Therapy - Methods & Clinical Development 2018 11, 1-8DOI: (10.1016/j.omtm.2018.08.007) Copyright © 2018 The Authors Terms and Conditions