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• W2754949897 abstract "Recombinant biopharmaceuticals can induce generation of anti-drug antibodies, which could potentially neutralize therapeutic drug activity. In this report, we describe development and validation of a cell-based assay for detection of neutralizing antibodies (Nab) against insulin and insulin analogues. In order to achieve clinically meaningful sensitivity the method used an early signalling event, insulin induced insulin receptor phosphorylation as the endpoint. Percentage insulin receptor phosphorylation in cell lysates was measured using ECL based ELISA. Presence of neutralizing antibodies (Nab) in samples will inhibit insulin induced receptor phosphorylation and consequently lead to a reduction in the percentage of phosphorylated insulin receptor. Additionally, usage of human insulin receptor overexpressing recombinant CHO cell line further improved the assay sensitivity by reducing the fixed drug (EC50) concentration used for induction of receptor phosphorylation. To ensure adequate free drug tolerance a pre-treatment step was introduced, where serum samples underwent acid dissociation and charcoal extraction before drug incubation. In order to distinguish ADA positive samples containing true Nab from samples containing non-antibody phosphorylation inhibitory serum factors, a confirmatory tier was integrated based on immunodepletion using protein AGL mix. Assay parameters including determination of screening and confirmatory cut-points, intra and inter assay precision, selectivity, specificity and stability were assessed during validation in accordance with recent regulatory guidelines and white papers. The advantage of selecting insulin receptor phosphorylation as assay endpoint made the assay capable of detecting Nab against insulin and insulin analogues." @default.
• W2754949897 created "2017-09-25" @default.
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• W2754949897 date "2018-01-01" @default.
• W2754949897 modified "2023-10-15" @default.
• W2754949897 title "Development and validation of a cell based assay for the detection of neutralizing antibodies against recombinant insulins" @default.
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• W2754949897 doi "https://doi.org/10.1016/j.jim.2017.09.004" @default.
• W2754949897 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/28935478" @default.
• W2754949897 hasPublicationYear "2018" @default.
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