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FRI0194 Use of gloresponsetm NF-κB-RE-LUC2P HEK293 cells to monitor drug and anti-drug antibody levels in serum
  1. B Hernandez-Breijo1,
  2. LY Bravo Gallego2,
  3. A Martinez-Feito1,
  4. A Jochems1,
  5. E Olariaga1,
  6. J Cheng3,
  7. L Delaurière4,
  8. F Fan3,
  9. T Schagat3,
  10. C Plasencia1,
  11. A Mezcua2,
  12. P Nozal2,
  13. A Balsa1,
  14. D Pascual-Salcedo1
  1. 1Immuno-Rheumatology research group
  2. 2Immunology, University Hospital la Paz, Madrid, Spain
  3. 3Promega Corporation, Madison (WI), United States
  4. 4Promega Europe Training and Applications Laboratory (PETAL), Promega Corporation, Charbonnières-les-Bains, France

Abstract

Background Rheumatoid Arthritis (RA) is often treated with anti-TNFα such as infliximab (Ifx) which in a long-term treatment can lead to the development of anti-Ifx antibodies (ATI), resulting in an interference with the drug activity. The investigation of the bioactivity of the circulating drug and antibodies present in patients sera with inflammatory diseases will allow to harmonize the different published data using both bioassays as well as immunoassays.

Objectives To evaluate the ability of the Promega bioassay in the quantification of both Ifx and ATI in serum from RA patients.

To compare the bioassay performance between capture- and bridging-ELISA.

Methods Serum Ifx-trough levels were determined in 50 samples from patients with RA. To measure Ifx, the bioassay uses GloResponse™ NF-κB-RE-luc2P HEK293 cell line, which responses to TNFα resulting in modulation of NF-κB activity (Promega Corp., Madison, USA). This is an ease on performing reporter assay (add-mix-read) that detects the serum Ifx by inhibition of the luminiscent signal due to TNFα. The inhibition of the Ifx-driven luminescent signal is reverted by the presence of ATI in the serum. ATI concentration is determined using a standard curve obtained with a serial dilution of a serum sample known to have ATI. A relative luminescent unit (RLU) is arbitrarily defined. The immunoassay for Ifx (ELISA) captures recombinant TNFα, via a monoclonal antibody that does not interfere with the serum Ifx binding to TNFα. The reaction is developed with a biotinylated anti-idiotype antibody1 (Cut-off for Ifx: 0.050 μg/ml). The ELISA to detect ATI takes advantage of the bivalency of most IgG subclasses, allowing the antibodies present in serum to bridge between Ifx-coated onto the plate with biotinylated Ifx in fluid phase1 (Cut-off for ATI 50 AU/ml).

Results Addition of TNFα on GloResponse cells induces the production of luciferase resulting in an increase of the luminescent signal in a dose-response. Addition of Ifx on GloResponse cells +TNFα decreases the production of luciferase and therefore the luminescent signal falls (r2=0.99). Addition of anti-Ifx (in relative units) on GloResponse cells +TNFα + Ifx restore the production of luciferase resulting in an increase of the luminescent signal (r2=0.98).

Serum Ifx concentrations were sorted into 3 groups (low, medium and high following ELISA levels). Similar number of positive Ifx results was found (30 samples, 55% by ELISA vs 28 samples, 52% by bioassay; κ=0.92; p=0.56) along with a significant Pearson correlation (r=0.8) between methods. The bioassay to measure ATI activity showed lower sensitivity than ELISA (9/31, 29% by ELISA vs 4/31, 13% by bioassay, κ=0.53, p=0.22) with a slightly less correlation for ATI levels by both methods (r=0.26).

Conclusions Bioassay using GloResponse NF-κB cell line can be performed to monitor therapeutic drug of TNFα blocker and are useful to detect the presence of anti-drug antibody in serum samples treated with anti-TNFα antibodies. Comparison with ELISA method shows in most of the case the same data.

References

  1. Pascual-Salcedo D. et al, Rheumatology, 2011.

References

Disclosure of Interest None declared

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