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Development and validation of cell-based luciferase reporter gene assays for measuring neutralizing anti-drug antibodies against interferon beta

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  1. An IgY-based immunoassay to evaluate the biomarker potential of the Tannerella forsythia virulence factor karilysin in human saliva

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  2. The use of synthetic peptides for detection of anti-citrullinated protein antibodies in rheumatoid arthritis

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  3. A novel antihuman C3d monoclonal antibody with specificity to the C3d complement split product

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  4. Efficient evaluation of humoral immune responses by the use of serum pools

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  1. Klinisk Neurologi og Neurokirurgi

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  2. Multiple sclerosis genomic map implicates peripheral immune cells and microglia in susceptibility

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  3. Serum and cerebrospinal fluid neurofilament levels predict longitudinal atrophy of the cervical spinal cord in progressive MS

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  4. Illegal cannabis use is common among Danes with multiple sclerosis

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Neutralizing anti-drug antibodies (NAbs) against therapeutic interferon beta (IFNβ) in people with multiple sclerosis (MS) are measured with cell-based bioassays. The aim of this study was to redevelop and validate two luciferase reporter-gene bioassays, LUC and iLite, using a cut-point approach to identify NAb positive samples. Such an approach is favored by the pharmaceutical industry and governmental regulatory agencies as it has a clear statistical basis and overcomes the limitations of the current assays based on the Kawade principle. The work was conducted following the latest assay guidelines. The assays were re-developed and validated as part of the "Anti-Biopharmaceutical Immunization: Prediction and analysis of clinical relevance to minimize the risk" (ABIRISK) consortium and involved a joint collaboration between four academic laboratories and two pharmaceutical companies. The LUC assay was validated at Innsbruck Medical University (LUCIMU) and at Rigshospitalet (LUCRH) Copenhagen, and the iLite assay at Karolinska Institutet, Stockholm. For both assays, the optimal serum sample concentration in relation to sensitivity and recovery was 2.5% (v/v) in assay media. A Shapiro-Wilk test indicated a normal distribution for the majority of runs, allowing a parametric approach for cut-point calculation to be used, where NAb positive samples could be identified with 95% confidence. An analysis of means and variances indicated that a floating cut-point should be used for all assays. The assays demonstrated acceptable sensitivity for being cell-based assays, with a confirmed limit of detection in neat serum of 1519 ng/mL for LUCIMU, 814 ng/mL for LUCRH, and 320 ng/mL for iLite. Use of the validated cut-point assay, in comparison with the previously used Kawade method, identified 14% more NAb positive samples. In conclusion, implementation of the cut-point design resulted in increased sensitivity to detect NAbs. However, the clinical significance of these low positive titers needs to be further evaluated.

Original languageEnglish
JournalJournal of Immunological Methods
Volume430
Pages (from-to)1-9
Number of pages9
ISSN0022-1759
DOIs
Publication statusPublished - Mar 2016

    Research areas

  • Antibodies, Neutralizing, Biological Assay, Cell Line, Tumor, Enzyme-Linked Immunosorbent Assay, Genes, Reporter, Humans, Interferon-beta, Luciferases, Multiple Sclerosis, Neutralization Tests, Sensitivity and Specificity, Journal Article, Research Support, Non-U.S. Gov't, Validation Studies

ID: 49730220