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The development and validation of an immunoassay for the measurement of anti-thymidine phosphorylase antibodies in mouse and dog sera

Gasson, C; Levene, M; Bax, BE (2013) The development and validation of an immunoassay for the measurement of anti-thymidine phosphorylase antibodies in mouse and dog sera. JOURNAL OF PHARMACEUTICAL AND BIOMEDICAL ANALYSIS, 72. 16 - 24 (9). ISSN 0731-7085 https://doi.org/10.1016/j.jpba.2012.09.009
SGUL Authors: Bax, Bridget Elizabeth

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%.

Item Type: Article
Additional Information: PubMed ID: 23146222
Keywords: Animals, Antibodies, Calibration, Dogs, Immunoassay, Mice, Mice, Inbred BALB C, Sensitivity and Specificity, Thymidine Phosphorylase, Science & Technology, Physical Sciences, Life Sciences & Biomedicine, Chemistry, Analytical, Pharmacology & Pharmacy, Chemistry, Assay validation, Bridging immunoassay, Enzyme replacement therapy, MNGIE, Thymidine phosphorylase, ADENOSINE-DEAMINASE, BIOTECHNOLOGY PRODUCTS, ALPHA-GLUCOSIDASE, HOST ANTIBODIES, POMPE-DISEASE, DEFICIENCY, THERAPY, RECOMMENDATIONS, DRUGS, Assay validation, Bridging immunoassay, Enzyme replacement therapy, MNGIE, Thymidine phosphorylase
SGUL Research Institute / Research Centre: Academic Structure > Molecular and Clinical Sciences Research Institute (MCS)
Academic Structure > Molecular and Clinical Sciences Research Institute (MCS) > Cell Sciences (INCCCS)
Journal or Publication Title: JOURNAL OF PHARMACEUTICAL AND BIOMEDICAL ANALYSIS
ISSN: 0731-7085
Related URLs:
Dates:
DateEvent
18 January 2013Published
Web of Science ID: WOS:000311819100003
URI: http://sgultest.da.ulcc.ac.uk/id/eprint/102268
Publisher's version: https://doi.org/10.1016/j.jpba.2012.09.009

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