title: The development and validation of an immunoassay for the measurement of anti-thymidine phosphorylase antibodies in mouse and dog sera.
abstract: Erythrocyte encapsulated thymidine phosphorylase (EE-TP) is under development as an enzyme replacement therapy for mitochondrial neurogastrointestinal encephalomyopathy (MNGIE), a fatal metabolic disorder resulting from an inherited deficiency of the enzyme thymidine phosphorylase. We report here the development and validation of a sensitive electrochemiluminescent (ECL) bridging immunoassay to support Good Laboratory Practice (GLP)-compliant preclinical safety studies of EE-TP in the mouse and dog. Affinity-purified rabbit anti-E. coli thymidine phosphorylase (TP) antibody was used as a calibrator standard with an effective working range of 2.5-7500 ng/mL. The minimum required dilution (MRD) for both mouse and dog sera was 1:10. The mean analytical recoveries for anti-TP antibodies spiked into serum at 70 ng/mL and 7000 ng/mL were 117.9% and 93.2%, respectively for mouse, and 112.0% and 104.3%, respectively for dog. The intra-assay precision (coefficient of variation, CV) ranged between 1.1% and 8.0% in mouse serum, and 1.9% and 2.5% in dog serum. Inter-assay precision ranged between -1.6% and 6.7% in mouse serum, and -13.0% and -2.5% in dog serum. Assay cut-point/screening cut-point correction factors were 201.37 and 44.4, respectively for mouse and dog sera. For future analysis of positive test samples, less than 37.12% (mouse) and 31.41% (dog) inhibition of the assay signal in the confirmation assay will confer anti-TP antibody specificity. Assay drift and hook effects (prozone) were not observed. The intra-assay and inter-assay accuracy for robustness were within ±20%.
authors.affiliation: Huntingdon Research Centre, Woolley Road, Alconbury, Huntingdon, Cambridgeshire, PE28 4HS, United Kingdom.
authors.forename: Charlotte
authors.initials: C
authors.lastname: Gasson
authors.forename: Michelle
authors.initials: M
authors.lastname: Levene
authors.forename: Bridget E
authors.initials: BE
authors.lastname: Bax
mesh_terms: D000818:Animals; D000906:Antibodies / Q000097:blood* / Q000737:chemistry / Q000276:immunology; D002138:Calibration; D004285:Dogs; D007118:Immunoassay / Q000379:methods* / Q000592:standards; D051379:Mice; D008807:Mice, Inbred BALB C; D012680:Sensitivity and Specificity; D013939:Thymidine Phosphorylase / Q000276:immunology*
chemical_list: D000906:Antibodies; D013939:Thymidine Phosphorylase