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  • Title: Development and optimization of a cell-based neutralizing antibody assay using a sample pre-treatment step to eliminate serum interference.
    Author: McCutcheon KM, Quarmby V, Song A.
    Journal: J Immunol Methods; 2010 Jun 30; 358(1-2):35-45. PubMed ID: 20361968.
    Abstract:
    We developed a neutralizing antibody assay (NAb assay), based upon the complement-dependent cytotoxicity (CDC) activity of a monoclonal human IgG(1) therapeutic (IgT), to characterize anti-therapeutic antibodies (ATA) in autoimmune patient serum. Neutralizing antibodies (NAb) were measured by a decrease in the extent of CDC mediated by 50 ng/mL IgT, on a lymphoblastoid cell line. A sample pre-treatment procedure, utilizing a Protein A/G resin to purify total immunoglobulins, was optimized for use in the NAb assay to eliminate the non-specific assay interferents observed in individual serum samples from rheumatoid arthritis patients. In some individuals, the addition of naïve serum to the assay completely inhibited CDC activity. After sample pre-treatment, the variability of the CDC response induced by IgT in individual serum samples from a drug-naïve RA population, tested over three days, was only 3%, irrespective of complement immune complexes or rheumatoid factor levels. The pre-treatment procedure was performed on samples and matrix controls as part of each assay. The NAb assay was able to recover and detect polyclonal ATA from human serum at a concentration of 0.25 microg/mL with pre-treatment. Dose-dependent neutralization of IgT was observed, however, a simple positive/negative reporting scheme was adopted. The NAb assay was found to have the desired properties of specificity, robustness, precision and recovery for validation to support the characterization of ATA in clinical samples.
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